Abstract:

The present invention provides methods of treating a cell proliferative
disorder, such as a cancer, by administering to a subject in need thereof
a therapeutically effective amount of a
pyrroloquinolinyl-pyrrole-2,5-dione compound or a
pyrroloquinolinyl-pyrrolidine-2,5-dione compound in combination with a
therapeutically effective amount of a second anti-proliferative agent.

Claims:

1. A method of treating a cell proliferative disorder, said method
comprising administering, to a subject in need thereof, a therapeutically
effective amount of a composition comprising
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione, or a pharmaceutically acceptable salt thereof,
or a prodrug or metabolite thereof, in combination with a therapeutically
effective amount of a second anti-proliferative agent, wherein said cell
proliferation disorder is treated.

2. The method of claim 1, wherein the second anti-proliferative agent is a
kinase inhibitor, an alkylating agent, an antibiotic, an anti-metabolite,
a detoxifying agent, an interferon, a polyclonal or monoclonal antibody,
a HER2 inhibitor, a histone deacetylase inhibitor, a hormone, a mitotic
inhibitor, an MTOR inhibitor, a taxane or taxane derivative, an aromatase
inhibitor, an anthracycline, a microtubule targeting drug, a
topoisomerase poison drug, or a cytidine analogue drug.

3. The method of claim 2, wherein the kinase inhibitor is a
serine/threonine kinase inhibitor.

4. The method of claim 2, wherein the kinase inhibitor is a tyrosine
kinase inhibitor.

20. The method of claim 19, wherein the cells have a constitutively
enhanced c-Met activity.

21. The method of claim 1, wherein the
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione is administered at a dose range between 0.1
mg/day to 10 g/day.

22. The method of claim 21, wherein the
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione is administered at a dose range between 0.1
mg/day to 5 g/day.

23. The method of claim 22, wherein the
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione is administered at a dose range between 10
mg/day to 1 g/day.

24. The method of claim 23, wherein the
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione is administered at a maximal daily dose of 720
mg.

25. The method of claim 24, wherein the
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione is administered at a dose of 360 mg, provided
twice a day.

26. The method of claim 1, wherein the composition comprising
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione, and the second anti-proliferative agent are
administered intravenously, orally or intraperitoneally.

27. The method of claim 1, wherein the second anti-proliferative agent is
administered simultaneously with, preceding administration of, or
following administration of the composition comprising
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione.

28. The method of claim 27, wherein the second anti-proliferative agent is
administered within 24 hours after the composition comprising
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione is administered.

29. The method of claim 1, wherein said composition further comprises one
or more pharmaceutically acceptable carriers or excipients.

30. The method of claim 1, wherein said second anti-proliferative agent
comprises one or more pharmaceutically acceptable carriers or excipients.

31. The method of claim 1, wherein the subject is a human.

32. A kit for the treatment of a cell proliferative disorder in a subject
comprising separate vials containing a composition comprising
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione, or a pharmaceutically acceptable salt thereof,
or a prodrug or metabolite thereof, and a second anti-proliferative
agent, with instructions for administering said composition and second
anti-proliferative agent.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application claims the benefit of U.S. Provisional Application
No. 61/152,138, filed Feb. 12, 2009 and U.S. Provisional Application No.
61/170,471, filed Apr. 17, 2009. The contents of each of these
applications are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002]Cancer is the second leading cause of death in the United States,
exceeded only by heart disease. (Cancer Facts and Figures 2004, American
Cancer Society, Inc.) Despite recent advances in cancer diagnosis and
treatment, surgery and radiotherapy may be curative if a cancer is found
early, but current drug therapies for metastatic disease are mostly
palliative and seldom offer a long-term cure. Even with new
chemotherapies entering the market, the need continues for new drugs
effective in monotherapy or in combination with existing agents as first
line therapy, and as second and third line therapies in treatment of
resistant tumors.

[0003]Cancer cells are by definition heterogeneous. For example, within a
single tissue or cell type, multiple mutational `mechanisms` may lead to
the development of cancer. As such, heterogeneity frequently exists
between cancer cells taken from tumors of the same tissue and same
histiotype that have originated in different individuals. Frequently
observed mutational `mechanisms` associated with some cancers may differ
between one tissue type and another (e.g., frequently observed mutational
`mechanisms` leading to colon cancer may differ from frequently observed
`mechanisms` leading to leukemias). It is therefore often difficult to
predict whether a particular cancer will respond to a particular
chemotherapeutic agent. (Cancer Medicine, 5th Edition, Bast et al. eds.,
B.C. Decker Inc., Hamilton, Ontario)

[0004]Breast cancer is the most frequently diagnosed non-skin cancer in
women, and ranks second among cancer deaths in women, after lung cancer.
(Cancer Facts and Figures 2004, American Cancer Society, Inc.) Current
treatment options for breast cancer include surgery, radiotherapy, and
chemotherapy/hormone therapy with agents such as tamoxifen, aromatase
inhibitors, HERCEPTIN® (trastuzumab), TAXOL® (paclitaxel),
cyclophosphamide, methotrexate, doxorubicin (Adriamycin®), and
5-fluorouracil (5-FU). Despite improvements in cancer diagnostics and
therapeutics, breast cancer incidence rates have continued to increase
since 1980. In 2004, about 215,000 new cases of breast cancer are
expected in women, and about 1,450 new cases of breast cancer are
expected in men. Id. Accordingly, new compounds and methods for treating
breast cancer are needed.

[0005]Components of cellular signal transduction pathways that regulate
the growth and differentiation of normal cells can, when dysregulated,
lead to the development of cellular proliferative disorders and cancer.
Mutations in cellular signaling proteins may cause such proteins to
become expressed or activated at inappropriate levels or at inappropriate
times during the cell cycle, which in turn may lead to uncontrolled
cellular growth or changes in cell-cell attachment properties. For
example, dysregulation of receptor tyrosine kinases by mutation, gene
rearrangement, gene amplification, and overexpression of both receptor
and ligand has been implicated in the development and progression of
human cancers.

[0006]The c-Met receptor tyrosine kinase is the only known high-affinity
receptor for hepatocyte growth factor (HGF), also known as scatter
factor. Binding of HGF to the c-Met extracellular ligand-binding domain
results in receptor multimerization and phosphorylation of multiple
tyrosine residues in the intracellular portion of c-Met. Activation of
c-Met results in the binding and phosphorylation of adaptor proteins such
as Gab-1, Grb-2, Shc, and c-Cbl, and subsequent activation of signal
transducers such as PI3K, PLC-γ, STATs, ERK1 and 2 and FAK. c-Met
and HGF are dysregulated in human cancers, and may contribute to
dysregulation of cell growth, tumor cell dissemination, and tumor
invasion during disease progression and metastasis. (See, e.g., Journal
of Clinical Investigation 109: 863-867 (2002) and Cancer Cell pp 5-6 Jul.
2004) c-Met and HGF are highly expressed relative to surrounding tissue
in numerous cancers, and their expression correlates with poor patient
prognosis. (See, e.g., Journal of Cellular Biochemistry 86: 665-677
(2002); Int. J. Cancer (Pred. Oncol.) 74: 301-309 (1997); Clinical Cancer
Research 9: 1480-1488 (2003); and Cancer Research 62: 589-596 (2002))
Without intending to be bound by theory, c-Met and HGF may protect tumors
against cell death induced by DNA damaging agents, and as such may
contribute to chemoresistance and radioresistance of tumors. Without
intending to be limited by any theory, inhibitors of c-Met may be useful
as therapeutic agents in the treatment of proliferative disorders
including breast cancer. (See, e.g., Cancer and Metastasis Reviews 22:
309-325 (2003))

[0007]The references cited herein are not admitted to be prior art to the
claimed invention.

SUMMARY OF THE INVENTION

[0008]The present invention provides a method of treating a cell
proliferative disorder, the method comprising administering to a subject
in need thereof a therapeutically effective amount of a compound of
formula III, IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptable
salt thereof, or a prodrug or metabolite thereof, with one or more
pharmaceutically acceptable carriers or excipients, alone, or in
combination with a therapeutically effective amount of a second
anti-proliferative agent, with one or more pharmaceutically acceptable
carriers or excipients, wherein the cell proliferation disorder is
treated.

[0010]The second anti-proliferative agent can be a a kinase inhibitor, an
alkylating agent, an antibiotic, an anti-metabolite, a detoxifying agent,
an interferon, a polyclonal or monoclonal antibody, a HER2 inhibitor, a
histone deacetylase inhibitor, a hormone, a mitotic inhibitor, an MTOR
inhibitor, a taxane or taxane derivative, an aromatase inhibitor, an
anthracycline, a microtubule targeting drug, a topoisomerase poison drug,
or a cytidine analogue drug. Preferably, the kinase inhibitor is
serine/threonine kinase inhibitor or a tyrosine kinase inhibitor.
Preferred kinase inhibitors include, but are not limited to, sorafenib,
sunitinib, erlotinib, imatinib, and gefitinib. Preferred alkylating
agents include, but are not limited to, cisplatin or carboplatin.
Preferred anti-metabolites include, but are not limited to, gemcitabine,
fluorouracil (5-FU), TS-1 or capecitabine. Preferred mitotic inhibitors
include, but are not limited to, camptothecin or irinotecan. Preferred
taxane or taxane derivatives include, but are not limited to, paclitaxel
or docetaxel.

[0012]Cells with a proliferative disorder can contain DNA encoding c-Met.
Alternatively, or in addition, cells with a proliferative disorder have a
constitutively enhanced c-Met activity. Preferably, the cell
proliferative disease is cancer, and particularly those cancers which
express c-Met at high levels or express active c-Met. Thus, the present
invention provides a method of treating cell proliferative disorders
where the cells express c-Met at high levels or express active c-Met. The
present invention further provides a method of treating a cell
proliferative disorder comprising selectively modulating an activity of
c-Met, without significantly inhibiting the activity of Protein Kinase C.

[0013]Preferably, the subject is a mammal. More preferably, the subject is
a human.

[0014]Preferably, the compound of formula III, IIIa, IVa, IVb, Va, or Vb
of the methods described herein is
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and is administered at a dose of 360 mg,
provided twice a day. Alternatively, the composition is administered at a
maximal daily dose of 720 mg.

[0015]Preferably, the
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and the second anti-proliferative agent are
administered intravenously, orally or intraperitoneally. The second
anti-proliferative agent can be administered simultaneously with,
preceding administration of, or following administration of the
composition comprising
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione. Preferably, the second anti-proliferative agent
is administered within 24 hours after the composition comprising
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione is administered.

[0016]The present invention also provides a kit for the treatment of a
cell proliferative disorder in a subject comprising separate vials
containing a composition comprising
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione, or a pharmaceutically acceptable salt thereof,
or a prodrug or metabolite thereof, and a second anti-proliferative
agent, with instructions for administering said composition and second
anti-proliferative agent.

[0017]Preferably, the subject is a mammal. More preferably, the subject is
a human.

[0018]Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. In the specification,
the singular forms also include the plural unless the context clearly
dictates otherwise. Although methods and materials similar or equivalent
to those described herein can be used in the practice or testing of the
present invention, suitable methods and materials are described below.
All publications, patent applications, patents and other references
mentioned herein are incorporated by reference. The references cited
herein are not admitted to be prior art to the claimed invention. In the
case of conflict, the present specification, including definitions, will
control. In addition, the materials, methods and examples are
illustrative only and are not intended to be limiting.

[0019]Other features and advantages of the invention will be apparent from
the following detailed description and claims.

[0022]FIG. 3 is a graph showing the anti-proliferative effect of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and sorafenib in the NCI-H522 NSCLC xenograft
model.

[0023]FIG. 4 is a graph showing the combinatorial anti-proliferative
effect of (-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(-
1H-indol-3-yl)pyrrolidine-2,5-dione in combination with sorafenib and
sunitinib on various cancer cell lines.

[0024]FIG. 5 is an illustration showing the combinatorial
anti-proliferative effect of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione in combination with sorafenib and sunitinib on
various cancer cell lines.

[0025]FIG. 6 is a graph showing the anti-proliferative effect of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione in combination with erlotinib in a NCI-H441
human lung tumor xenograft model.

[0026]FIG. 7 is a graph showing the anti-proliferative effect of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione in combination with gefitinib in a NCI-H441
human lung tumor xenograft model.

[0027]FIG. 8 is a graph showing the dose response curves for the
combinatinorial treatment of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and gemcitabine in pancreatic cell lines.

[0028]FIG. 9 is a graph depicting the volume of a MKN-45 human gastric
tumor in a xenograft model as a proportion of its initial volume (V/Vo)
following treatment with various doses of vehicle (control),
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione (Agent A), Docetaxel (DTX), or a combination
thereof. # indicates p<0.05 vs. DTX treatment alone by a student's
t-test. ## indicates p<0.01 vs. DTX treatment alone by a student's
t-test.

[0029]FIG. 10 is a graph depicting the volume of a Hsc-39 human gastric
tumor in a xenograft model as a proportion of its initial volume (V/Vo)
following treatment with various doses of vehicle,
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione (Agent A), Docetaxel (DTX), or a combination
thereof. # indicates p<0.05 vs. DTX treatment alone by a stuendent's
t-test. ## indicates p<0.01 vs. DTX treatment alone by a student's
t-test.

[0030]FIG. 11 is a graph depicting the volume of a MKN-45 human gastric
tumor in a xenograft model as a proportion of its initial volume (V/Vo)
following treatment with various doses of vehicle,
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione (Agent A), 5-FU, or a combination thereof. #
indicates p<0.05 vs. 5-FU treatment alone. * indicates p<0.05 vs.
Agent A treatment alone by a student's t-test.

[0031]FIG. 12 is a graph depicting the volume of a MKN-45 human gastric
tumor in a xenograft model as a proportion of its initial volume (V/Vo)
following treatment with various doses of vehicle,
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione (Agent A), TS-1, or a combination thereof. #
indicates p<0.05 vs. TS-1 treatment alone. * indicates p<0.05 vs.
Agent A treatment alone by a student's t-test.

[0032]FIG. 13 is a graph depicting the volume of a MKN-45 human gastric
tumor in a xenograft model as a proportion of its initial volume (V/Vo)
following treatment with various doses of vehicle,
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione (Agent A), Capecitabine, or a combination
thereof. # indicates p<0.05 vs. Capecitabine treatment alone. *
indicates p<0.05 vs. Agent A treatment alone by a student's t-test.

[0033]FIG. 14 is a graph depicting the volume of a MKN-45 human gastric
tumor in a xenograft model as a proportion of its initial volume (V/Vo)
following treatment with various doses of vehicle,
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione (Agent A), CDDP, or a combination thereof.

DETAILED DESCRIPTION OF THE INVENTION

1. Methods of Treatment

[0034]The present invention provides methods of treating a cell
proliferative disorder, the method comprising administering to a subject
in need thereof a therapeutically effective amount of a compound of
formula III, IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptable
salt thereof, or a prodrug or metabolite thereof, with one or more
pharmaceutically acceptable carriers or excipients, alone, or in
combination with a therapeutically effective amount of a second
anti-proliferative agent, with one or more pharmaceutically acceptable
carriers or excipients, wherein the cell proliferation disorder is
treated.

[0035]The present invention provides a pharmaceutical composition for
treating a cell proliferative disorder, comprising a combination of (a) a
therapeutically effective amount of a compound of formula III, IIIa, IVa,
IVb, Va, or Vb, or a pharmaceutically acceptable salt thereof, or a
prodrug or metabolite thereof, alone, or in combination with (b) a
therapeutically effective amount of a second anti-proliferative agent.
One or more pharmaceutically acceptable carriers or excipients is (are)
optionally included in the composition.

[0036]A second anti-proliferative agent is preferably a second
chemotherapeutic agent.

[0037]The cell proliferative disorder can be a precancerous condition or
cancer. The cell proliferative disorder can be a hematologic tumor or
malignancy, or a solid tumor (or tumors). This method of treating cancer
include a reduction in tumor size. Alternatively, or in addition, the
cancer is metastatic cancer and this method of treatment includes
inhibition of metastatic cancer cell invasion.

[0038]The second chemotherapeutic agent (also referred to as an
anti-neoplastic agent or anti-proliferative agent) can be an alkylating
agent; an antibiotic; an anti-metabolite; a detoxifying agent; an
interferon; a polyclonal or monoclonal antibody; an EGFR inhibitor; a
HER2 inhibitor; a histone deacetylase inhibitor; a hormone; a mitotic
inhibitor; an MTOR inhibitor; a multi-kinase inhibitor; a
serine/threonine kinase inhibitor; a tyrosine kinase inhibitors; a
VEGF/VEGFR inhibitor; a taxane or taxane derivative, an aromatase
inhibitor, an anthracycline, a microtubule targeting drug, a
topoisomerase poison drug, an inhibitor of a molecular target or enzyme
(e.g., a kinase inhibitor), a cytidine analogue drug or any
chemotherapeutic, anti-neoplastic or anti-proliferative agent listed in
www.cancer.org/docroot/cdg/cdg--0.asp.

[0063]In another aspect, the second chemotherapeutic agent can be a
cytokine such as G-CSF (granulocyte colony stimulating factor). In
another aspect, a compound of formula III, IIIa, IVa, IVb, Va, or Vb, or
a pharmaceutically acceptable salt, prodrug, metabolite, analog or
derivative thereof, may be administered in combination with radiation
therapy. Radiation therapy can also be administered in combination with a
compound of formula III, IIIa, IVa, IVb, Va, or Vb and another
chemotherapeutic agent described herein as part of a multiple agent
therapy. In yet another aspect, a compound of formula III, IIIa, IVa,
IVb, Va, or Vb, or a pharmaceutically acceptable salt, prodrug,
metabolite, analog or derivative thereof, may be administered in
combination with standard chemotherapy combinations such as, but not
restricted to, CMF (cyclophosphamide, methotrexate and 5-fluorouracil),
CAF (cyclophosphamide, adriamycin and 5-fluorouracil), AC (adriamycin and
cyclophosphamide), FEC (5-fluorouracil, epirubicin, and
cyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide, and
paclitaxel), rituximab, Xeloda (capecitabine), Cisplatin (CDDP),
Carboplatin, TS-1 (tegafur, gimestat and otastat potassium at a molar
ratio of 1:0.4:1), Camptothecin-11 (CPT-11, Irinotecan or Camptosar®)
or CMFP (cyclophosphamide, methotrexate, 5-fluorouracil and prednisone).

[0064]In preferred embodiments, a compound of formula III, IIIa, IVa, IVb,
Va, or Vb, or a pharmaceutically acceptable salt, prodrug, metabolite,
polymorph or solvate thereof, may be administered with an inhibitor of an
enzyme, such as a receptor or non-receptor kinase. Receptor and
non-receptor kinases of the invention are, for example, tyrosine kinases
or serine/threonine kinases. Kinase inhibitors of the invention are small
molecules, polynucleic acids, polypeptides, or antibodies.

[0065]Preferred combinatorial therapies include, but are not limited to,
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione administered in combination with erlotinib,
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione administered in combination with sorafenib,
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione administered in combination with sunitinib;
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione administered in combination with capecitabine;
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione administered in combination with carboplatin and
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione administered in combination with cisplatin. In
certain embodiments, a subject or patient receives a combination of
erlotinib, administered as 150 mg once daily, in combination with
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione, administered as 360 mg twice daily. In another
embodiment, a subject or patient receives a combination of sorafenib,
administered as 200 mg twice daily, in combination with
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione, administered as 360 mg twice daily. Preferred
dosage forms for
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione include, but are not limited to, a capsule and a
tablet.

[0067]A compound of formula III, IIIa, IVa, IVb, Va, or Vb of the present
invention, or a pharmaceutically acceptable salt, prodrug, metabolite,
analog or derivative thereof, can be incorporated into pharmaceutical
compositions suitable for administration. Such compositions typically
comprise the compound (i.e. including the active compound), and a
pharmaceutically acceptable excipient or carrier. As used herein,
"pharmaceutically acceptable excipient" or "pharmaceutically acceptable
carrier" is intended to include any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like, compatible with pharmaceutical
administration. Suitable carriers are described in the most recent
edition of Remington's Pharmaceutical Sciences, a standard reference text
in the field. Preferred examples of such carriers or diluents include,
but are not limited to, water, saline, ringer's solutions, dextrose
solution, and 5% human serum albumin.

[0068]Pharmaceutically acceptable carriers include solid carriers such as
lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia,
magnesium stearate, stearic acid and the like. Exemplary liquid carriers
include syrup, peanut oil, olive oil, water and the like. Similarly, the
carrier or diluent may include time-delay material known in the art, such
as glyceryl monostearate or glyceryl distearate, alone or with a wax,
ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate or the
like. Other fillers, excipients, flavorants, and other additives such as
are known in the art may also be included in a pharmaceutical composition
according to this invention. Liposomes and non-aqueous vehicles such as
fixed oils may also be used. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.

[0069]In one aspect, a compound of formula III, IIIa, IVa, IVb, Va, or Vb,
or a pharmaceutically acceptable salt, prodrug, metabolite, analog or
derivative thereof, is administered in a suitable dosage form prepared by
combining a therapeutically effective amount (e.g., an efficacious level
sufficient to achieve the desired therapeutic effect through inhibition
of tumor growth, killing of tumor cells, treatment or prevention of cell
proliferative disorders, etc.) of a compound of formula III, Ma, IVa,
IVb, Va, or Vb, or a pharmaceutically acceptable salt, prodrug,
metabolite, analog or derivative thereof, (as an active ingredient) with
standard pharmaceutical carriers or diluents according to conventional
procedures (i.e., by producing a pharmaceutical composition of the
invention). These procedures may involve mixing, granulating, and
compressing or dissolving the ingredients as appropriate to attain the
desired preparation.

[0070]As used herein, a "subject" can be any mammal, e.g., a human, a
primate, mouse, rat, dog, cat, cow, horse, pig, sheep, goat, camel. In a
preferred aspect, the subject is a human.

[0071]As used herein, a "subject in need thereof" is a subject having a
cell proliferative disorder, or a subject having an increased risk of
developing a cell proliferative disorder relative to the population at
large. In one aspect, a subject in need thereof has a precancerous
condition. In a preferred aspect, a subject in need thereof has cancer.

[0072]As used herein, the term "cell proliferative disorder" refers to
conditions in which unregulated or abnormal growth, or both, of cells can
lead to the development of an unwanted condition or disease, which may or
may not be cancerous. Exemplary cell proliferative disorders of the
invention encompass a variety of conditions wherein cell division is
deregulated. Exemplary cell proliferative disorder include, but are not
limited to, neoplasms, benign tumors, malignant tumors, pre-cancerous
conditions, in situ tumors, encapsulated tumors, metastatic tumors,
liquid tumors, solid tumors, immunological tumors, hematological tumors,
cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividing
cells. The term "rapidly dividing cell" as used herein is defined as any
cell that divides at a rate that exceeds or is greater than what is
expected or observed among neighboring or juxtaposed cells within the
same tissue. A cell proliferative disorder includes a precancer or a
precancerous condition. A cell proliferative disorder includes cancer.
Preferably, the methods provided herein are used to treat or alleviate a
symptom of cancer. The term "cancer" includes solid tumors, as well as,
hematologic tumors and/or malignancies. A "precancer cell" or
"precancerous cell" is a cell manifesting a cell proliferative disorder
that is a precancer or a precancerous condition. A "cancer cell" or
"cancerous cell" is a cell manifesting a cell proliferative disorder that
is a cancer. Any reproducible means of measurement may be used to
identify cancer cells or precancerous cells. Cancer cells or precancerous
cells can be identified by histological typing or grading of a tissue
sample (e.g., a biopsy sample). Cancer cells or precancerous cells can be
identified through the use of appropriate molecular markers.

[0075]A "cell proliferative disorder of the hematologic system" is a cell
proliferative disorder involving cells of the hematologic system. In one
aspect, a cell proliferative disorder of the hematologic system includes
lymphoma, leukemia, myeloid neoplasms, mast cell neoplasms,
myelodysplasia, benign monoclonal gammopathy, lymphomatoid
granulomatosis, lymphomatoid papulosis, polycythemia vera, chronic
myelocytic leukemia, agnogenic myeloid metaplasia, and essential
thrombocythemia. In another aspect, a cell proliferative disorder of the
hematologic system includes hyperplasia, dysplasia, and metaplasia of
cells of the hematologic system. In a preferred aspect, compositions of
the present invention may be used to treat a cancer selected from the
group consisting of a hematologic cancer of the present invention or a
hematologic cell proliferative disorder of the present invention. In one
aspect, a hematologic cancer of the present invention includes multiple
myeloma, lymphoma (including Hodgkin's lymphoma, non-Hodgkin's lymphoma,
childhood lymphomas, and lymphomas of lymphocytic and cutaneous origin),
leukemia (including childhood leukemia, hairy-cell leukemia, acute
lymphocytic leukemia, acute myelocytic leukemia, chronic lymphocytic
leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, and
mast cell leukemia), myeloid neoplasms and mast cell neoplasms.

[0076]A "cell proliferative disorder of the lung" is a cell proliferative
disorder involving cells of the lung. In one aspect, cell proliferative
disorders of the lung include all forms of cell proliferative disorders
affecting lung cells. In one aspect, cell proliferative disorders of the
lung include lung cancer, a precancer or precancerous condition of the
lung, benign growths or lesions of the lung, and malignant growths or
lesions of the lung, and metastatic lesions in tissue and organs in the
body other than the lung. In a preferred aspect, compositions of the
present invention may be used to treat lung cancer or cell proliferative
disorders of the lung. In one aspect, lung cancer includes all forms of
cancer of the lung. In another aspect, lung cancer includes malignant
lung neoplasms, carcinoma in situ, typical carcinoid tumors, and atypical
carcinoid tumors. In another aspect, lung cancer includes small cell lung
cancer ("SCLC"), non-small cell lung cancer ("NSCLC"), squamous cell
carcinoma, adenocarcinoma, small cell carcinoma, large cell carcinoma,
adenosquamous cell carcinoma, and mesothelioma. In another aspect, lung
cancer includes "scar carcinoma," bronchioalveolar carcinoma, giant cell
carcinoma, spindle cell carcinoma, and large cell neuroendocrine
carcinoma. In another aspect, lung cancer includes lung neoplasms having
histologic and ultrastructual heterogeneity (e.g., mixed cell types).

[0077]In one aspect, cell proliferative disorders of the lung include all
forms of cell proliferative disorders affecting lung cells. In one
aspect, cell proliferative disorders of the lung include lung cancer,
precancerous conditions of the lung. In one aspect, cell proliferative
disorders of the lung include hyperplasia, metaplasia, and dysplasia of
the lung. In another aspect, cell proliferative disorders of the lung
include asbestos-induced hyperplasia, squamous metaplasia, and benign
reactive mesothelial metaplasia. In another aspect, cell proliferative
disorders of the lung include replacement of columnar epithelium with
stratified squamous epithelium, and mucosal dysplasia. In another aspect,
individuals exposed to inhaled injurious environmental agents such as
cigarette smoke and asbestos may be at increased risk for developing cell
proliferative disorders of the lung. In another aspect, prior lung
diseases that may predispose individuals to development of cell
proliferative disorders of the lung include chronic interstitial lung
disease, necrotizing pulmonary disease, scleroderma, rheumatoid disease,
sarcoidosis, interstitial pneumonitis, tuberculosis, repeated pneumonias,
idiopathic pulmonary fibrosis, granulomata, asbestosis, fibrosing
alveolitis, and Hodgkin's disease.

[0078]A "cell proliferative disorder of the colon" is a cell proliferative
disorder involving cells of the colon. In a preferred aspect, the cell
proliferative disorder of the colon is colon cancer. In a preferred
aspect, compositions of the present invention may be used to treat colon
cancer or cell proliferative disorders of the colon. In one aspect, colon
cancer includes all forms of cancer of the colon. In another aspect,
colon cancer includes sporadic and hereditary colon cancers. In another
aspect, colon cancer includes malignant colon neoplasms, carcinoma in
situ, typical carcinoid tumors, and atypical carcinoid tumors. In another
aspect, colon cancer includes adenocarcinoma, squamous cell carcinoma,
and adenosquamous cell carcinoma. In another aspect, colon cancer is
associated with a hereditary syndrome selected from the group consisting
of hereditary nonpolyposis colorectal cancer, familial adenomatous
polyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndrome
and juvenile polyposis. In another aspect, colon cancer is caused by a
hereditary syndrome selected from the group consisting of hereditary
nonpolyposis colorectal cancer, familial adenomatous polyposis, Gardner's
syndrome, Peutz-Jeghers syndrome, Turcot's syndrome and juvenile
polyposis.

[0079]In one aspect, cell proliferative disorders of the colon include all
forms of cell proliferative disorders affecting colon cells. In one
aspect, cell proliferative disorders of the colon include colon cancer,
precancerous conditions of the colon, adenomatous polyps of the colon and
metachronous lesions of the colon. In one aspect, a cell proliferative
disorder of the colon includes adenoma. In one aspect, cell proliferative
disorders of the colon are characterized by hyperplasia, metaplasia, and
dysplasia of the colon. In another aspect, prior colon diseases that may
predispose individuals to development of cell proliferative disorders of
the colon include prior colon cancer. In another aspect, current disease
that may predispose individuals to development of cell proliferative
disorders of the colon include Crohn's disease and ulcerative colitis. In
one aspect, a cell proliferative disorder of the colon is associated with
a mutation in a gene selected from the group consisting of p53, ras, FAP
and DCC. In another aspect, an individual has an elevated risk of
developing a cell proliferative disorder of the colon due to the presence
of a mutation in a gene selected from the group consisting of p53, ras,
FAP and DCC.

[0080]A "cell proliferative disorder of the prostate" is a cell
proliferative disorder involving cells of the prostate. In one aspect,
cell proliferative disorders of the prostate include all forms of cell
proliferative disorders affecting prostate cells. In one aspect, cell
proliferative disorders of the prostate include prostate cancer, a
precancer or precancerous condition of the prostate, benign growths or
lesions of the prostate, and malignant growths or lesions of the
prostate, and metastatic lesions in tissue and organs in the body other
than the prostate. In another aspect, cell proliferative disorders of the
prostate include hyperplasia, metaplasia, and dysplasia of the prostate.

[0081]A "cell proliferative disorder of the skin" is a cell proliferative
disorder involving cells of the skin. In one aspect, cell proliferative
disorders of the skin include all forms of cell proliferative disorders
affecting skin cells. In one aspect, cell proliferative disorders of the
skin include a precancer or precancerous condition of the skin, benign
growths or lesions of the skin, melanoma, malignant melanoma and other
malignant growths or lesions of the skin, and metastatic lesions in
tissue and organs in the body other than the skin. In another aspect,
cell proliferative disorders of the skin include hyperplasia, metaplasia,
psoriasis, and dysplasia of the skin.

[0082]A "cell proliferative disorder of the ovary" is a cell proliferative
disorder involving cells of the ovary. In one aspect, cell proliferative
disorders of the ovary include all forms of cell proliferative disorders
affecting cells of the ovary. In one aspect, cell proliferative disorders
of the ovary include a precancer or precancerous condition of the ovary,
benign growths or lesions of the ovary, ovarian cancer, malignant growths
or lesions of the ovary, and metastatic lesions in tissue and organs in
the body other than the ovary. In another aspect, cell proliferative
disorders of the ovary include hyperplasia, metaplasia, and dysplasia of
cells of the ovary.

[0083]A "cell proliferative disorder of the breast" is a cell
proliferative disorder involving cells of the breast. In one aspect, cell
proliferative disorders of the breast include all forms of cell
proliferative disorders affecting breast cells. In one aspect, cell
proliferative disorders of the breast include breast cancer, a precancer
or precancerous condition of the breast, benign growths or lesions of the
breast, and malignant growths or lesions of the breast, and metastatic
lesions in tissue and organs in the body other than the breast. In
another aspect, cell proliferative disorders of the breast include
hyperplasia, metaplasia, and dysplasia of the breast.

[0084]In one aspect, a cell proliferative disorder of the breast is a
precancerous condition of the breast. In one aspect, compositions of the
present invention may be used to treat a precancerous condition of the
breast. In one aspect, a precancerous condition of the breast includes
atypical hyperplasia of the breast, ductal carcinoma in situ (DCIS),
intraductal carcinoma, lobular carcinoma in situ (LCIS), lobular
neoplasia, and stage 0 or grade 0 growth or lesion of the breast (e.g.,
stage 0 or grade 0 breast cancer, or carcinoma in situ). In another
aspect, a precancerous condition of the breast has been staged according
to the TNM classification scheme as accepted by the American Joint
Committee on Cancer (AJCC), where the primary tumor (T) has been assigned
a stage of T0 or T is; and where the regional lymph nodes (N) have been
assigned a stage of NO; and where distant metastasis (M) has been
assigned a stage of MO.

[0085]In a preferred aspect, the cell proliferative disorder of the breast
is breast cancer. In a preferred aspect, compositions of the present
invention may be used to treat breast cancer. In one aspect, breast
cancer includes all forms of cancer of the breast. In one aspect, breast
cancer includes primary epithelial breast cancers. In another aspect,
breast cancer includes cancers in which the breast is involved by other
tumors such as lymphoma, sarcoma or melanoma. In another aspect, breast
cancer includes carcinoma of the breast, ductal carcinoma of the breast,
lobular carcinoma of the breast, undifferentiated carcinoma of the
breast, cystosarcoma phyllodes of the breast, angiosarcoma of the breast,
and primary lymphoma of the breast. In one aspect, breast cancer includes
Stage I, II, IIIA, IIIB, IIIC and IV breast cancer. In one aspect, ductal
carcinoma of the breast includes invasive carcinoma, invasive carcinoma
in situ with predominant intraductal component, inflammatory breast
cancer, and a ductal carcinoma of the breast with a histologic type
selected from the group consisting of comedo, mucinous (colloid),
medullary, medullary with lymphcytic infiltrate, papillary, scirrhous,
and tubular. In one aspect, lobular carcinoma of the breast includes
invasive lobular carcinoma with predominant in situ component, invasive
lobular carcinoma, and infiltrating lobular carcinoma. In one aspect,
breast cancer includes Paget's disease, Paget's disease with intraductal
carcinoma, and Paget's disease with invasive ductal carcinoma. In another
aspect, breast cancer includes breast neoplasms having histologic and
ultrastructual heterogeneity (e.g., mixed cell types).

[0086]In a preferred aspect, a compound of formula III, IIIa, IVa, IVb,
Va, or Vb may be used to treat breast cancer. In one aspect, a breast
cancer that is to be treated includes familial breast cancer. In another
aspect, a breast cancer that is to be treated includes sporadic breast
cancer. In one aspect, a breast cancer that is to be treated has arisen
in a male subject. In one aspect, a breast cancer that is to be treated
has arisen in a female subject. In one aspect, a breast cancer that is to
be treated has arisen in a premenopausal female subject or a
postmenopausal female subject. In one aspect, a breast cancer that is to
be treated has arisen in a subject equal to or older than 30 years old,
or a subject younger than 30 years old. In one aspect, a breast cancer
that is to be treated has arisen in a subject equal to or older than 50
years old, or a subject younger than 50 years old. In one aspect, a
breast cancer that is to be treated has arisen in a subject equal to or
older than 70 years old, or a subject younger than 70 years old.

[0087]In one aspect, a breast cancer that is to be treated has been typed
to identify a familial or spontaneous mutation in BRCA1, BRCA2, or p53.
In one aspect, a breast cancer that is to be treated has been typed as
having a HER2/neu gene amplification, as overexpressing HER2/neu, or as
having a low, intermediate or high level of HER2/neu expression. In
another aspect, a breast cancer that is to be treated has been typed for
a marker selected from the group consisting of estrogen receptor (ER),
progesterone receptor (PR), human epidermal growth factor receptor-2,
Ki-67, CA15-3, CA 27-29, and c-Met. In one aspect, a breast cancer that
is to be treated has been typed as ER-unknown, ER-rich or ER-poor. In
another aspect, a breast cancer that is to be treated has been typed as
ER-negative or ER-positive. ER-typing of a breast cancer may be performed
by any reproducible means. In a preferred aspect, ER-typing of a breast
cancer may be performed as set forth in Onkologie 27: 175-179 (2004). In
one aspect, a breast cancer that is to be treated has been typed as
PR-unknown, PR-rich or PR-poor. In another aspect, a breast cancer that
is to be treated has been typed as PR-negative or PR-positive. In another
aspect, a breast cancer that is to be treated has been typed as receptor
positive or receptor negative. In one aspect, a breast cancer that is to
be treated has been typed as being associated with elevated blood levels
of CA 15-3, or CA 27-29, or both.

[0088]In one aspect, a breast cancer that is to be treated includes a
localized tumor of the breast. In one aspect, a breast cancer that is to
be treated includes a tumor of the breast that is associated with a
negative sentinel lymph node (SLN) biopsy. In one aspect, a breast cancer
that is to be treated includes a tumor of the breast that is associated
with a positive sentinel lymph node (SLN) biopsy. In another aspect, a
breast cancer that is to be treated includes a tumor of the breast that
is associated with one or more positive auxiliary lymph nodes, where the
auxiliary lymph nodes have been staged by any applicable method. In
another aspect, a breast cancer that is to be treated includes a tumor of
the breast that has been typed as having nodal negative status (e.g.,
node-negative) or nodal positive status (e.g., node-positive). In another
aspect, a breast cancer that is to be treated includes a tumor of the
breast that has metastasized to other locations in the body. In one
aspect, a breast cancer that is to be treated is classified as having
metastasized to a location selected from the group consisting of bone,
lung, liver, or brain. In another aspect a breast cancer that is to be
treated is classified according to a characteristic selected from the
group consisting of metastatic, localized, regional, local-regional,
locally advanced, distant, multicentric, bilateral, ipsilateral,
contralateral, newly diagnosed, recurrent, and inoperable.

[0089]In one aspect, a compound of formula III, IIIa, IVa, IVb, Va, or Vb
may be used to treat or prevent a cell proliferative disorder of the
breast, or to treat or prevent breast cancer, in a subject having an
increased risk of developing breast cancer relative to the population at
large. In one aspect, a subject with an increased risk of developing
breast cancer relative to the population at large is a female subject
with a family history or personal history of breast cancer. In another
aspect, a subject with an increased risk of developing breast cancer
relative to the population at large is a female subject having a
germ-line or spontaneous mutation in BRCA1 or BRCA2, or both. In one
aspect, a subject with an increased risk of developing breast cancer
relative to the population at large is a female subject with a family
history of breast cancer and a germ-line or spontaneous mutation in BRCA1
or BRCA2, or both. In another aspect, a subject with an increased risk of
developing breast cancer relative to the population at large is a female
who is greater than 30 years old, greater than 40 years old, greater than
50 years old, greater than 60 years old, greater than 70 years old,
greater than 80 years old, or greater than 90 years old. In one aspect, a
subject with an increased risk of developing breast cancer relative to
the population at large is a subject with atypical hyperplasia of the
breast, ductal carcinoma in situ (DCIS), intraductal carcinoma, lobular
carcinoma in situ (LCIS), lobular neoplasia, or a stage 0 growth or
lesion of the breast (e.g., stage 0 or grade 0 breast cancer, or
carcinoma in situ).

[0090]In another aspect, a breast cancer that is to be treated has been
histologically graded according to the Scarff-Bloom-Richardson system,
wherein a breast tumor has been assigned a mitosis count score of 1, 2,
or 3; a nuclear pleiomorphism score of 1, 2, or 3; a tubule formation
score of 1, 2, or 3; and a total Scarff-Bloom-Richardson score of between
3 and 9. In another aspect, a breast cancer that is to be treated has
been assigned a tumor grade according to the International Consensus
Panel on the Treatment of Breast Cancer selected from the group
consisting of grade 1, grade 1-2, grade 2, grade 2-3, or grade 3.

[0091]In one aspect, a cancer that is to be treated has been staged
according to the American Joint Committee on Cancer (AJCC) TNM
classification system, where the tumor (T) has been assigned a stage of
TX, T1, T1mic, T1a, T1b, T1c, T2, T3, T4, T4a, T4b, T4c, or T4d; and
where the regional lymph nodes (N) have been assigned a stage of NX, N0,
N1, N2, N2a, N2b, N3, N3a, N3b, or N3c; and where distant metastasis (M)
has been assigned a stage of MX, MO, or Ml. In another aspect, a cancer
that is to be treated has been staged according to an American Joint
Committee on Cancer (AJCC) classification as Stage I, Stage IIA, Stage
IIB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV. In another aspect,
a cancer that is to be treated has been assigned a grade according to an
AJCC classification as Grade GX (e.g., grade cannot be assessed), Grade
1, Grade 2, Grade 3 or Grade 4. In another aspect, a cancer that is to be
treated has been staged according to an AJCC pathologic classification
(pN) of pNX, pN0, PN0 (I-), PN0 (I+), PN0 (mol-), PN0 (mol+), PN1,
PN1(mi), PN1a, PN1b, PN1c, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.

[0092]In one aspect, a cancer that is to be treated includes a tumor that
has been determined to be less than or equal to about 2 centimeters in
diameter. In another aspect, a cancer that is to be treated includes a
tumor that has been determined to be from about 2 to about 5 centimeters
in diameter. In another aspect, a cancer that is to be treated includes a
tumor that has been determined to be greater than or equal to about 3
centimeters in diameter. In another aspect, a cancer that is to be
treated includes a tumor that has been determined to be greater than 5
centimeters in diameter. In another aspect, a cancer that is to be
treated is classified by microscopic appearance as well differentiated,
moderately differentiated, poorly differentiated, or undifferentiated. In
another aspect, a cancer that is to be treated is classified by
microscopic appearance with respect to mitosis count (e.g., amount of
cell division) or nuclear pleiomorphism (e.g., change in cells). In
another aspect, a cancer that is to be treated is classified by
microscopic appearance as being associated with areas of necrosis (e.g.,
areas of dying or degenerating cells). In one aspect, a cancer that is to
be treated is classified as having an abnormal karyotype, having an
abnormal number of chromosomes, or having one or more chromosomes that
are abnormal in appearance. In one aspect, a cancer that is to be treated
is classified as being aneuploid, triploid, tetraploid, or as having an
altered ploidy. In one aspect, a cancer that is to be treated is
classified as having a chromosomal translocation, or a deletion or
duplication of an entire chromosome, or a region of deletion, duplication
or amplification of a portion of a chromosome.

[0093]In one aspect, a cancer that is to be treated is evaluated by DNA
cytometry, flow cytometry, or image cytometry. In one aspect, a cancer
that is to be treated has been typed as having 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, or 90% of cells in the synthesis stage of cell division
(e.g., in S phase of cell division). In one aspect, a cancer that is to
be treated has been typed as having a low S-phase fraction or a high
S-phase fraction.

[0094]As used herein, a "normal cell" is a cell that cannot be classified
as part of a "cell proliferative disorder." In one aspect, a normal cell
lacks unregulated or abnormal growth, or both, that can lead to the
development of an unwanted condition or disease. Preferably, a normal
cell possesses normally functioning cell cycle checkpoint control
mechanisms.

[0095]As used herein, "contacting a cell" refers to a condition in which a
compound or other composition of matter is in direct contact with a cell,
or is close enough to induce a desired biological effect in a cell.

[0096]As used herein, "candidate compound" refers to a compound of formula
III, IIIa, IVa, IVb, Va, or Vb that has been or will be tested in one or
more in vitro or in vivo biological assays, in order to determine if that
compound is likely to elicit a desired biological or medical response in
a cell, tissue, system, animal or human that is being sought by a
researcher or clinician. In one aspect, a candidate compound is a
compound of formula III or IIIa; in another aspect, a candidate compound
is a compound of formula IVa, IVb, Va, or Vb. In a preferred aspect, the
biological or medical response is treatment of cancer. In another aspect,
the biological or medical response is treatment or prevention of a cell
proliferative disorder. In one aspect, in vitro or in vivo biological
assays include, but are not limited to, enzymatic activity assays,
electrophoretic mobility shift assays, reporter gene assays, in vitro
cell viability assays, and the assays.

[0097]As used herein, "monotherapy" refers to administration of a single
active or therapeutic compound to a subject in need thereof. Preferably,
monotherapy will involve administration of a therapeutically effective
amount of an active compound. For example, cancer monotherapy with
(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol--
3-yl)pyrrolidine-2,5-dione comprises administration of a therapeutically
effective amount of
(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol--
3-yl)pyrrolidine-2,5-dione, or a pharmaceutically acceptable salt,
prodrug, metabolite, analog or derivative thereof, to a subject in need
of treatment of cancer. Monotherapy may be contrasted with combination
therapy, in which a combination of multiple active compounds is
administered, preferably with each component of the combination present
in a therapeutically effective amount. In one aspect, montherapy with a
compound of formula III, IIIa, IVa, IVb, Va, or Vb is more effective than
combination therapy in inducing a desired biological effect.
Monotherapeutic effectiveness of the compounds of formula III, IIIa, IVa,
IVb, Va, or Vb is shown in PCT Publication No. WO 2006/086484.

[0098]As used herein, "treating" describes the management and care of a
patient for the purpose of combating a disease, condition, or disorder
and includes decreasing or alleviating the symptoms or complications, or
eliminating the disease, condition or disorder.

[0099]As used herein, "preventing" describes stopping the onset of the
symptoms or complications of the disease, condition or disorder.

[0100]In one aspect, treating cancer results in a reduction in size of a
tumor. A reduction in size of a tumor may also be referred to as "tumor
regression." Preferably, after treatment, tumor size is reduced by 5% or
greater relative to its size prior to treatment; more preferably, tumor
size is reduced by 10% or greater; more preferably, reduced by 20% or
greater; more preferably, reduced by 30% or greater; more preferably,
reduced by 40% or greater; even more preferably, reduced by 50% or
greater; and most preferably, reduced by greater than 75% or greater.
Size of a tumor may be measured by any reproducible means of measurement.
In a preferred aspect, size of a tumor may be measured as a diameter of
the tumor.

[0101]In another aspect, treating cancer results in a reduction in tumor
volume. Preferably, after treatment, tumor volume is reduced by 5% or
greater relative to its size prior to treatment; more preferably, tumor
volume is reduced by 10% or greater; more preferably, reduced by 20% or
greater; more preferably, reduced by 30% or greater; more preferably,
reduced by 40% or greater; even more preferably, reduced by 50% or
greater; and most preferably, reduced by greater than 75% or greater.
Tumor volume may be measured by any reproducible means of measurement.

[0102]In another aspect, treating cancer results in a decrease in number
of tumors. Preferably, after treatment, tumor number is reduced by 5% or
greater relative to number prior to treatment; more preferably, tumor
number is reduced by 10% or greater; more preferably, reduced by 20% or
greater; more preferably, reduced by 30% or greater; more preferably,
reduced by 40% or greater; even more preferably, reduced by 50% or
greater; and most preferably, reduced by greater than 75%. Number of
tumors may be measured by any reproducible means of measurement. In a
preferred aspect, number of tumors may be measured by counting tumors
visible to the naked eye or at a specified magnification. In a preferred
aspect, the specified magnification is 2×, 3×, 4×,
5×, 10×, or 50×.

[0103]In another aspect, treating cancer results in a decrease in number
of metastatic lesions in other tissues or organs distant from the primary
tumor site. Preferably, after treatment, the number of metastatic lesions
is reduced by 5% or greater relative to number prior to treatment; more
preferably, the number of metastatic lesions is reduced by 10% or
greater; more preferably, reduced by 20% or greater; more preferably,
reduced by 30% or greater; more preferably, reduced by 40% or greater;
even more preferably, reduced by 50% or greater; and most preferably,
reduced by greater than 75%. The number of metastatic lesions may be
measured by any reproducible means of measurement. In a preferred aspect,
the number of metastatic lesions may be measured by counting metastatic
lesions visible to the naked eye or at a specified magnification. In a
preferred aspect, the specified magnification is 2×, 3×,
4×, 5×, 10×, or 50×.

[0104]In another aspect, treating cancer results in an increase in average
survival time of a population of treated subjects in comparison to a
population receiving carrier alone. Preferably, the average survival time
is increased by more than 30 days; more preferably, by more than 60 days;
more preferably, by more than 90 days; and most preferably, by more than
120 days. An increase in average survival time of a population may be
measured by any reproducible means. In a preferred aspect, an increase in
average survival time of a population may be measured, for example, by
calculating for a population the average length of survival following
initiation of treatment with an active compound. In another preferred
aspect, an increase in average survival time of a population may also be
measured, for example, by calculating for a population the average length
of survival following completion of a first round of treatment with an
active compound.

[0105]In another aspect, treating cancer results in an increase in average
survival time of a population of treated subjects in comparison to a
population of untreated subjects. Preferably, the average survival time
is increased by more than 30 days; more preferably, by more than 60 days;
more preferably, by more than 90 days; and most preferably, by more than
120 days. An increase in average survival time of a population may be
measured by any reproducible means. In a preferred aspect, an increase in
average survival time of a population may be measured, for example, by
calculating for a population the average length of survival following
initiation of treatment with an active compound. In another preferred
aspect, an increase in average survival time of a population may also be
measured, for example, by calculating for a population the average length
of survival following completion of a first round of treatment with an
active compound.

[0106]In another aspect, treating cancer results in increase in average
survival time of a population of treated subjects in comparison to a
population receiving monotherapy with a drug that is not a compound of
formula III, IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptable
salt, prodrug, metabolite, analog or derivative thereof. Preferably, the
average survival time is increased by more than 30 days; more preferably,
by more than 60 days; more preferably, by more than 90 days; and most
preferably, by more than 120 days. An increase in average survival time
of a population may be measured by any reproducible means. In a preferred
aspect, an increase in average survival time of a population may be
measured, for example, by calculating for a population the average length
of survival following initiation of treatment with an active compound. In
another preferred aspect, an increase in average survival time of a
population may also be measured, for example, by calculating for a
population the average length of survival following completion of a first
round of treatment with an active compound.

[0107]In another aspect, treating cancer results in a decrease in the
mortality rate of a population of treated subjects in comparison to a
population receiving carrier alone. In another aspect, treating cancer
results in a decrease in the mortality rate of a population of treated
subjects in comparison to an untreated population. In a further aspect,
treating cancer results a decrease in the mortality rate of a population
of treated subjects in comparison to a population receiving monotherapy
with a drug that is not a compound of formula III, IIIa, IVa, IVb, Va, or
Vb, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or
derivative thereof. Preferably, the mortality rate is decreased by more
than 2%; more preferably, by more than 5%; more preferably, by more than
10%; and most preferably, by more than 25%. In a preferred aspect, a
decrease in the mortality rate of a population of treated subjects may be
measured by any reproducible means. In another preferred aspect, a
decrease in the mortality rate of a population may be measured, for
example, by calculating for a population the average number of
disease-related deaths per unit time following initiation of treatment
with an active compound. In another preferred aspect, a decrease in the
mortality rate of a population may also be measured, for example, by
calculating for a population the average number of disease-related deaths
per unit time following completion of a first round of treatment with an
active compound.

[0108]In another aspect, treating cancer results in a decrease in tumor
growth rate. Preferably, after treatment, tumor growth rate is reduced by
at least 5% relative to number prior to treatment; more preferably, tumor
growth rate is reduced by at least 10%; more preferably, reduced by at
least 20%; more preferably, reduced by at least 30%; more preferably,
reduced by at least 40%; more preferably, reduced by at least 50%; even
more preferably, reduced by at least 50%; and most preferably, reduced by
at least 75%. Tumor growth rate may be measured by any reproducible means
of measurement. In a preferred aspect, tumor growth rate is measured
according to a change in tumor diameter per unit time.

[0109]In another aspect, treating cancer results in a decrease in tumor
regrowth. Preferably, after treatment, tumor regrowth is less than 5%;
more preferably, tumor regrowth is less than 10%; more preferably, less
than 20%; more preferably, less than 30%; more preferably, less than 40%;
more preferably, less than 50%; even more preferably, less than 50%; and
most preferably, less than 75%. Tumor regrowth may be measured by any
reproducible means of measurement. In a preferred aspect, tumor regrowth
is measured, for example, by measuring an increase in the diameter of a
tumor after a prior tumor shrinkage that followed treatment. In another
preferred aspect, a decrease in tumor regrowth is indicated by failure of
tumors to reoccur after treatment has stopped.

[0110]In another aspect, treating or preventing a cell proliferative
disorder results in a reduction in the rate of cellular proliferation.
Preferably, after treatment, the rate of cellular proliferation is
reduced by at least 5%; more preferably, by at least 10%; more
preferably, by at least 20%; more preferably, by at least 30%; more
preferably, by at least 40%; more preferably, by at least 50%; even more
preferably, by at least 50%; and most preferably, by at least 75%. The
rate of cellular proliferation may be measured by any reproducible means
of measurement. In a preferred aspect, the rate of cellular proliferation
is measured, for example, by measuring the number of dividing cells in a
tissue sample per unit time.

[0111]In another aspect, treating or preventing a cell proliferative
disorder results in a reduction in the proportion of proliferating cells.
Preferably, after treatment, the proportion of proliferating cells is
reduced by at least 5%; more preferably, by at least 10%; more
preferably, by at least 20%; more preferably, by at least 30%; more
preferably, by at least 40%; more preferably, by at least 50%; even more
preferably, by at least 50%; and most preferably, by at least 75%. The
proportion of proliferating cells may be measured by any reproducible
means of measurement. In a preferred aspect, the proportion of
proliferating cells is measured, for example, by quantifying the number
of dividing cells relative to the number of nondividing cells in a tissue
sample. In another preferred aspect, the proportion of proliferating
cells is equivalent to the mitotic index.

[0112]In another aspect, treating or preventing a cell proliferative
disorder results in a decrease in size of an area or zone of cellular
proliferation. Preferably, after treatment, size of an area or zone of
cellular proliferation is reduced by at least 5% relative to its size
prior to treatment; more preferably, reduced by at least 10%; more
preferably, reduced by at least 20%; more preferably, reduced by at least
30%; more preferably, reduced by at least 40%; more preferably, reduced
by at least 50%; even more preferably, reduced by at least 50%; and most
preferably, reduced by at least 75%. Size of an area or zone of cellular
proliferation may be measured by any reproducible means of measurement.
In a preferred aspect, size of an area or zone of cellular proliferation
may be measured as a diameter or width of an area or zone of cellular
proliferation.

[0113]In another aspect, treating or preventing a cell proliferative
disorder results in a decrease in the number or proportion of cells
having an abnormal appearance or morphology. Preferably, after treatment,
the number of cells having an abnormal morphology is reduced by at least
5% relative to its size prior to treatment; more preferably, reduced by
at least 10%; more preferably, reduced by at least 20%; more preferably,
reduced by at least 30%; more preferably, reduced by at least 40%; more
preferably, reduced by at least 50%; even more preferably, reduced by at
least 50%; and most preferably, reduced by at least 75%. An abnormal
cellular appearance or morphology may be measured by any reproducible
means of measurement. In one aspect, an abnormal cellular morphology is
measured by microscopy, e.g., using an inverted tissue culture
microscope. In one aspect, an abnormal cellular morphology takes the form
of nuclear pleiomorphism.

[0114]As used herein, the term "selectively" means tending to occur at a
higher frequency in one population than in another population. In one
aspect, the compared populations are cell populations. In a preferred
aspect, a compound of formula III, IIIa, IVa, IVb, Va, or Vb, or a
pharmaceutically acceptable salt, prodrug, metabolite, analog or
derivative thereof, acts selectively on a cancer or precancerous cell but
not on a normal cell. In another preferred aspect, a compound of formula
III, IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptable salt,
prodrug, metabolite, analog or derivative thereof, acts selectively to
modulate one molecular target (e.g., c-Met) but does not significantly
modulate another molecular target (e.g., Protein Kinase C). In another
preferred aspect, the invention provides a method for selectively
inhibiting the activity of an enzyme, such as a kinase. Preferably, an
event occurs selectively in population A relative to population B if it
occurs greater than two times more frequently in population A as compared
to population B. More preferably, an event occurs selectively if it
occurs greater than five times more frequently in population A. More
preferably, an event occurs selectively if it occurs greater than ten
times more frequently in population A; more preferably, greater than
fifty times; even more preferably, greater than 100 times; and most
preferably, greater than 1000 times more frequently in population A as
compared to population B. For example, cell death would be said to occur
selectively in cancer cells if it occurred greater than twice as
frequently in cancer cells as compared to normal cells.

[0115]In a preferred aspect, a compound of formula III, IIIa, IVa, IVb,
Va, or Vb or a pharmaceutically acceptable salt, prodrug, metabolite,
analog or derivative thereof, modulates the activity of a molecular
target (e.g., c-Met). In one aspect, modulating refers to stimulating or
inhibiting an activity of a molecular target. Preferably, a compound of
formula III, IIIa, IVa, IVb, Va, or Vb modulates the activity of a
molecular target if it stimulates or inhibits the activity of the
molecular target by at least 2-fold relative to the activity of the
molecular target under the same conditions but lacking only the presence
of said compound. More preferably, a compound of formula III, IIIa, IVa,
IVb, Va, or Vb modulates the activity of a molecular target if it
stimulates or inhibits the activity of the molecular target by at least
5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least
100-fold relative to the activity of the molecular target under the same
conditions but lacking only the presence of said compound. The activity
of a molecular target may be measured by any reproducible means. The
activity of a molecular target may be measured in vitro or in vivo. For
example, the activity of a molecular target may be measured in vitro by
an enzymatic activity assay or a DNA binding assay, or the activity of a
molecular target may be measured in vivo by assaying for expression of a
reporter gene.

[0116]In one aspect, a compound of formula III, IIIa, IVa, IVb, Va, or Vb,
or a pharmaceutically acceptable salt, prodrug, metabolite, analog or
derivative thereof, does not significantly modulate the activity of a
molecular target if the addition of the compound does not stimulate or
inhibit the activity of the molecular target by greater than 10% relative
to the activity of the molecular target under the same conditions but
lacking only the presence of said compound. In a preferred aspect, a
compound of formula III, IIIa, IVa, IVb, Va, or Vb does not significantly
modulate the activity of Protein Kinase C.

[0117]As used herein, the term "isozyme selective" means preferential
inhibition or stimulation of a first isoform of an enzyme in comparison
to a second isoform of an enzyme (e.g., preferential inhibition or
stimulation of a kinase isozyme alpha in comparison to a kinase isozyme
beta). Preferably, a compound of formula III, IIIa, IVa, IVb, Va, or Vb
demonstrates a minimum of a four fold differential, preferably a ten fold
differential, more preferably a fifty fold differential, in the dosage
required to achieve a biological effect. Preferably, a compound of
formula III, IIIa, IVa, IVb, Va, or Vb demonstrates this differential
across the range of inhibition, and the differential is exemplified at
the IC50, i.e., a 50% inhibition, for a molecular target of
interest.

[0118]In a preferred embodiment, administering a compound of formula III,
IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptable salt,
prodrug, metabolite, analog or derivative thereof, to a cell or a subject
in need thereof results in modulation (i.e., stimulation or inhibition)
of an activity of c-Met. As used herein, an activity of c-Met refers to
any biological function or activity that is carried out by c-Met. For
example, a function of c-Met includes phosphorylation of downstream
target proteins. Other functions of c-Met include autophosphorylation,
binding of adaptor proteins such as Gab-1, Grb-2, Shc, SHP2 and c-Cbl,
and activation of signal transducers such as Ras, Src, PI3K, PLC-γ,
STATs, ERK1 and 2 and FAK. c-Met knockdown has been shown to inhibit
cancer cell growth in a cell-type-specific manner. MDA-MB-231, NCI-H661,
NCI-H441, MIA PaCa-2, HT29 and MKN-45 human cancer cells. c-Met knockdown
induces caspase-dependent apoptosis in a cell type-specific manner. Thus,
the present invention is directed to the treatment of cell proliferative
disorders where the cells express c-Met at high levels or express active
c-Met.

[0119]In a preferred embodiment, administering a compound of formula III,
IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptable salt,
prodrug, metabolite, analog or derivative thereof, to a cell or a subject
in need thereof results in modulation (i.e., stimulation or inhibition)
of an activity of ERK 1 or ERK 2, or both. As used herein, an activity of
ERK 1 or ERK 2 refers to any biological function or activity that is
carried out by ERK 1 or ERK 2. For example, a function of ERK 1 or ERK 2
includes phosphorylation of downstream target proteins.

[0120]In one aspect, activating refers to placing a composition of matter
(e.g., protein or nucleic acid) in a state suitable for carrying out a
desired biological function. In one aspect, a composition of matter
capable of being activated also has an unactivated state. In one aspect,
an activated composition of matter may have an inhibitory or stimulatory
biological function, or both.

[0121]In one aspect, elevation refers to an increase in a desired
biological activity of a composition of matter (e.g., a protein or a
nucleic acid). In one aspect, elevation may occur through an increase in
concentration of a composition of matter.

[0122]As used herein, "a cell cycle checkpoint pathway" refers to a
biochemical pathway that is involved in modulation of a cell cycle
checkpoint. A cell cycle checkpoint pathway may have stimulatory or
inhibitory effects, or both, on one or more functions comprising a cell
cycle checkpoint. A cell cycle checkpoint pathway is comprised of at
least two compositions of matter, preferably proteins, both of which
contribute to modulation of a cell cycle checkpoint. A cell cycle
checkpoint pathway may be activated through an activation of one or more
members of the cell cycle checkpoint pathway. Preferably, a cell cycle
checkpoint pathway is a biochemical signaling pathway.

[0123]As used herein, "cell cycle checkpoint regulator" refers to a
composition of matter that can function, at least in part, in modulation
of a cell cycle checkpoint. A cell cycle checkpoint regulator may have
stimulatory or inhibitory effects, or both, on one or more functions
comprising a cell cycle checkpoint. In one aspect, a cell cycle
checkpoint regulator is a protein. In another aspect, a cell cycle
checkpoint regulator is not a protein.

[0124]In one aspect, treating cancer or a cell proliferative disorder
results in cell death, and preferably, cell death results in a decrease
of at least 10% in number of cells in a population. More preferably, cell
death means a decrease of at least 20%; more preferably, a decrease of at
least 30%; more preferably, a decrease of at least 40%; more preferably,
a decrease of at least 50%; most preferably, a decrease of at least 75%.
Number of cells in a population may be measured by any reproducible
means. In one aspect, number of cells in a population is measured by
fluorescence activated cell sorting (FACS). In another aspect, number of
cells in a population is measured by immunofluorescence microscopy. In
another aspect, number of cells in a population is measured by light
microscopy. In another aspect, methods of measuring cell death are as
shown in Li et al., (2003) Proc Natl Acad Sci USA. 100(5): 2674-8. In an
aspect, cell death occurs by apoptosis.

[0125]In a preferred aspect, an effective amount of a compound of formula
III, IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptable salt,
prodrug, metabolite, analog or derivative thereof is not significantly
cytotoxic to normal cells. A therapeutically effective amount of a
compound is not significantly cytotoxic to normal cells if administration
of the compound in a therapeutically effective amount does not induce
cell death in greater than 10% of normal cells. A therapeutically
effective amount of a compound does not significantly affect the
viability of normal cells if administration of the compound in a
therapeutically effective amount does not induce cell death in greater
than 10% of normal cells. In an aspect, cell death occurs by apoptosis.

[0126]In one aspect, contacting a cell with a compound of formula III,
IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptable salt,
prodrug, metabolite, analog or derivative thereof, induces or activates
cell death selectively in cancer cells. Preferably, administering to a
subject in need thereof a compound of formula III, IIIa, IVa, IVb, Va, or
Vb, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or
derivative thereof, induces or activates cell death selectively in cancer
cells. In another aspect, contacting a cell with a compound of formula
III, IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptable salt,
prodrug, metabolite, analog or derivative thereof, induces cell death
selectively in one or more cells affected by a cell proliferative
disorder. Preferably, administering to a subject in need thereof a
compound of formula III, IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically
acceptable salt, prodrug, metabolite, analog or derivative thereof,
induces cell death selectively in one or more cells affected by a cell
proliferative disorder. In a preferred aspect, the present invention
relates to a method of treating or preventing cancer by administering a
compound of formula III, IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically
acceptable salt, prodrug, metabolite, analog or derivative thereof to a
subject in need thereof, where administration of the compound of formula
III, IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptable salt,
prodrug, metabolite, analog or derivative thereof results in one or more
of the following: accumulation of cells in G1 and/or S phase of the cell
cycle, cytotoxicity via cell death in cancer cells without a significant
amount of cell death in normal cells, antitumor activity in animals with
a therapeutic index of at least 2, and activation of a cell cycle
checkpoint. As used herein, "therapeutic index" is the maximum tolerated
dose divided by the efficacious dose. One skilled in the art may refer to
general reference texts for detailed descriptions of known techniques
discussed herein or equivalent techniques. These texts include Ausubel et
al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc.
(2005); Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed.),
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2000); Coligan et
al., Current Protocols in Immunology, John Wiley & Sons, N.Y.; Enna et
al., Current Protocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et
al., The Pharmacological Basis of Therapeutics (1975), Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 18th edition
(1990). These texts can, of course, also be referred to in making or
using an aspect of the invention.

[0129]R4 is independently selected from the group consisting of hydrogen,
--(C1-C6) alkyl, --CH2R7;

[0130]R5, R6 are independently selected from the group consisting of
hydrogen, and --(C1-C6) alkyl;

[0131]R7 is independently selected from the group consisting of
--O--P(═O)(OH)2, --O--P(═O)(--OH)(--O--(C1-C6)
alkyl), --O--P(═O)(--O--(C1-C6) alkyl)2,
--O--P(═O)(--OH) (--O--(CH2)-phenyl),
--O--P(═O)(--O--(CH2)-phenyl)2, a carboxylic acid group, an
amino carboxylic acid group and a peptide;

[0132]Q is selected from the group consisting of aryl, heteroaryl,
--O-aryl, --S-aryl, --O-heteroaryl, and --S-heteroaryl;

[0133]X is selected from the group consisting of --(CH2)--, --(NR8)-,
S, and O;

[0138]For the compound of formula Ma, Q is selected from the group
consisting of aryl, heteroaryl, --O-aryl, --S-aryl, --O-heteroaryl, and
--S-heteroaryl, provided that when R4 is hydrogen, or (C1-C4)
alkyl, Q is not 3-indolyl or substituted 3-indolyl.

[0141]R4 is independently selected from the group consisting of hydrogen,
--(C1-C6) alkyl, --CH2R7;

[0142]R5, R6 are independently selected from the group consisting of
hydrogen, and --(C1-C6) alkyl;

[0143]R7 is independently selected from the group consisting of
--O--P(═O)(OH)2, --O--P(═O)(--OH)(--O--(C1-C6)
alkyl), --O--P(═O)(--O--(C1-C6) alkyl)2,
--O--P(═O)(--OH) (--O--(CH2)-phenyl),
--O--P(═O)(--O--(CH2)-phenyl)2, a carboxylic acid group, an
amino carboxylic acid group and a peptide;

[0144]Q is selected from the group consisting of aryl, heteroaryl,
--O-aryl, --S-aryl, --O-heteroaryl, and --S-heteroaryl;

[0145]X is selected from the group consisting of --(CH2)--, --(NR8)-,
S, and O;

[0150]The term "alkyl" refers to radicals containing carbon and hydrogen,
without unsaturation. Alkyl radicals can be straight or branched.
Exemplary alkyl radicals include, without limitation, methyl, ethyl,
propyl, isopropyl, hexyl, t-butyl, sec-butyl and the like. Alkyl groups
may be denoted by a range, thus, for example, a (C1-C6) alkyl
group is an alkyl group having from one to six carbon atoms in the
straight or branched alkyl backbone. Substituted and unsubstituted alkyl
groups may independently be (C1-C5) alkyl, (C1-C6)
alkyl, (C1-C10) alkyl, (C3-C10) alkyl, or
(C5-C10) alkyl. Unless expressly stated, the term "alkyl" does
not include "cycloalkyl."

[0151]A "cycloalkyl" group refers to a cyclic alkyl group having the
indicated number of carbon atoms in the "ring portion," where the "ring
portion" may consist of one or more ring structures either as fused,
spiro, or bridged ring structures. For example, a C3 to C6
cycloalkyl group (e.g., (C3-C6) cycloalkyl) is a ring structure
having between 3 and 6 carbon atoms in the ring. When no range is given,
then cycloalkyl has between three and nine carbon atoms
((C3-C9) cycloalkyl) in the ring portion. Exemplary cycloalkyl
groups include, but are not limited to cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, and adamantyl. Preferred cycloalkyl
groups have three, four, five, six, seven, eight, nine, or from three to
nine carbon atoms in the ring structure.

[0152]The term substituted alkyl and substituted cycloalkyl, refer to
alkyl and cycloalkyl groups, as defined above, substituted with one or
more substituents independently selected from the group consisting of
fluorine, aryl, heteroaryl, --O--(C1-C6) alkyl, and --NR5R6,
where R5 and R6 are independently selected from the group consisting of
hydrogen and --(C1-C6) alkyl.

[0153]The term "aryl" refers to an aromatic carbocyclic group, having one,
two, or three aromatic rings. Exemplary aryl groups include, without
limitation, phenyl, naphthyl, and the like. Aryl groups include one, two,
or three aromatic rings structures fused with one or more additional
nonaromatic carbocyclic or heterocyclic rings having from 4-9 members.
Examples of fused aryl groups include benzocyclobutanyl, indanyl,
tetrahydronapthylenyl, 1,2,3,4-tetrahydrophenanthrenyl,
tetrahydroanthracenyl, 1,4-dihydro-1,4-methanonaphthalenyl,
benzodioxolyl.

[0154]The term "heteroaryl" refers to a heteroaromatic (heteroaryl) group
having one, two, or three aromatic rings containing from 1-4 heteroatoms
(such as nitrogen, sulfur, or oxygen) in the aromatic ring. Heteroaryl
groups include one, two, or three aromatic rings structures containing
from 1-4 heteroatoms fused with one or more additional nonaromatic rings
having from 4-9 members. Heteroaryl groups containing a single type of
heteroatom in the aromatic ring are denoted by the type of hetero atom
they contain, thus, nitrogen-containing heteroaryl, oxygen-containing
heteroaryl and sulfur-containing heteroaryl denote heteroaromatic groups
containing one or more nitrogen, oxygen or sulfur atoms respectively.
Exemplary heteroaryl groups include, without limitation, pyridyl,
pyrimidinyl, triazolyl, quinolyl, quinazolinyl, thiazolyl,
benzo[b]thiophenyl, furanyl, imidazolyl, indolyl, and the like.

[0155]The terms "heterocyclyl" or "heterocycle" refers to either saturated
or unsaturated, stable non-aromatic ring structures that may be fused,
spiro or bridged to form additional rings. Each heterocycle consists of
one or more carbon atoms and from one to four heteroatoms selected from
the group consisting of nitrogen, oxygen and sulfur. "Heterocyclyl" or
"heterocycle" include stable non-aromatic 3-7 membered monocyclic
heterocyclic ring structures and 8-11 membered bicyclic heterocyclic ring
structures. A heterocyclyl radical may be attached at any endocyclic
carbon or nitrogen atom that results in the creation of a stable
structure. Preferred heterocycles include 3-7 membered monocyclic
heterocycles (more preferably 5-7-membered monocyclic heterocycles) and
8-10 membered bicyclic heterocycles. Examples of such groups include
piperidinyl, piperazinyl, pyranyl, pyrrolidinyl, morpholinyl,
thiomorpholinyl, oxopiperidinyl, oxopyrrolidinyl, oxoazepinyl, azepinyl,
isoxozolyl, tetrahydropyranyl, tetrahydrofuranyl, dioxolyl, dioxinyl,
oxathiolyl, dithiolyl, sulfolanyl, dioxanyl, dioxolanyl,
tetahydrofurodihydrofuranyl, tetrahydropyranodihydro-furanyl,
dihydropyranyl, tetrahydrofurofuranyl, tetrahydropyranofuran,
quinuclidinyl (1-azabicyclo[2.2.2]octanyl) and tropanyl
(8-methyl-8-azabicyclo[3.2.1]octanyl).

[0157]For the purposes of the R7 substituent, the term "carboxylic acid
group" refers to a group of the form --O--C(═O)--(C1-C6)
alkyl, --O--C(═O)--(C3-C9) cycloalkyl, --O--C(═O)-aryl,
--O--C(═O)-heteroaryl, --O--C(═O)-heterocycle,
--O--C(═O)--(C1-C6) alkyl-aryl,
--O--C(═O)--(C1-C6) alkyl-heteroaryl, or
--O--C(═O)--(C1-C6) alkyl-heterocycle. Included in
"carboxylic acid group" are groups of the form
--O--C(═O)--(C1-C6) alkyl,
--O--C(═O)--(C3-C9) cycloalkyl, --O--C(═O)-aryl,
--O--C(═O)-heteroaryl, --O--C(═O)-heterocycle,
--O--C(═O)--(C1-C6) alkyl-aryl,
--O--C(═O)--(C1-C6) alkyl-heteroaryl, or
--O--C(═O)--(C1-C6) alkyl-heterocycle substituted with one
or more substituent independently selected from the group consisting of:
F, Cl, Br, I, --OH, --SH, --NR5R6, --(C1-C6) alkyl,
--(C1-C6) substituted alkyl, --(C3-C9) cycloalkyl,
--(C3-C9) substituted cycloalkyl, --O--(C1-C6) alkyl,
--O--(C1-C6) substituted alkyl, --S--(C1-C6) alkyl,
--O--(C3-C9) cycloalkyl, --O--(C3-C9) substituted
cycloalkyl, -aryl, --O-aryl, --O--(C1-C4) alkyl-aryl,
heteroaryl, heterocyclyl, --O--(C1-C4) alkyl-heterocycle,
--(S(═O)2)--(C1-C6) alkyl, --NH--C(═NH)--NH2
(i.e., guanido), --COOH, and --C(═O)--NR5R6, where R5 and R6 are
independently selected from the group consisting of hydrogen, and
--(C1-C6) alkyl. In addition, for the purposes of the R7
substituent the term "amino carboxylic acid group" refers to a carboxylic
acid group, including carboxylic acid groups substituted with one or more
of the above-stated substituents, which bears one or more independently
selected amino groups of the form --NR5R6 where R5 and R6 are
independently selected from the group consisting of hydrogen and
(C1-C6) alkyl.

[0159]For the purposes of the R7 substituent, the term "peptide" refers to
a dipeptide, tripeptide, tetrapeptide or pentapeptide, which release two,
three, four, or five amino or imino acids (e.g., proline) respectively
upon hydrolysis. For the purpose of R7, peptides are linked to the
remainder of the molecule through an ester linkage. In one embodiment,
peptides of R7 are comprised of alpha amino or imino acid, including but
not limited to alanine, arginine, asparagine, aspartic acid, cysteine,
glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, proline, serine, threonine,
tryptophan, tyrosine, valine or stereoisomers or racemic mixtures
thereof; and in a more preferred version of this embodiment, the carboxyl
group involved in the ester linkage is the carboxyl terminal COOH group
of the peptide, as opposed to a side chain carboxyl. In another
embodiment the of the invention, R7 is alpha amino or imino acid selected
from the group consisting of L-alanine, L-arginine, L-asparagine,
L-aspartic acid, L-cysteine, L-glutamine, L-glutamic acid, L-glycine,
L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,
L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,
L-tyrosine, and L-valine; and in a more preferred version of this
preferred embodiment, the carboxyl group involved in the ester linkage is
the carboxyl terminal COOH group of the peptide, as opposed to a side
chain carboxyl.

2.2. Preferred Compounds

[0160]Included in the preferred embodiments are compounds of formula III,
IIIa, IVa, IVb, Va, or Vb, wherein Q is selected from the group
consisting of aryl, heteroaryl, --O-aryl, --S-aryl, --O-heteroaryl, and
--S-heteroaryl, provided that Q is not 3-indolyl or a substituted
3-indolyl. In other preferred embodiments Q is selected from the group
consisting of aryl, heteroaryl, --O-aryl, --S-aryl, --O-heteroaryl, and
--S-heteroaryl, provided that when R4 is hydrogen, cycloalkyl, or alkyl,
Q is not 3-indolyl or a substituted 3-indolyl. In still other preferred
embodiments Q is selected from the group consisting of aryl, heteroaryl,
--O-aryl, --S-aryl, --O-heteroaryl, and --S-heteroaryl, provided that
when R4 is hydrogen, (C3-C4) cycloalkyl, or (C1-C4)
alkyl, Q is not 3-indolyl or substituted 3-indolyl. In another preferred
embodiment Q is 3-indolyl or a substituted 3-indolyl provided that R4 is
not hydrogen, cycloalkyl, or alkyl. In still another preferred embodiment
Q is 3-indolyl or a substituted 3-indolyl provided that R4 is not
hydrogen, (C3-C4) cycloalkyl, or (C1-C4) alkyl.

[0161]Other preferred embodiments include compounds of formula III, IIIa,
IVa, IVb, Va, or Vb where R4 is --CH2R7. These compounds may serve
as prodrug forms of the corresponding compounds of formula III, IIIa,
IVa, IVb, Va, or Vb where R4 is H. The prodrug form is cleaved by
hydrolysis to release the corresponding compound where R4 is H. The
hydrolysis may occur by enzymatic or nonenzymatic routes that produce the
corresponding hydroxymethylene derivative, which upon subsequent
hydrolysis, result in the release of compounds where R4 is H. In one such
preferred embodiment R4 is --CH2R7, where R7 is
--O--P(═O)(OH)2,
--O--P(═O)(--OH)(--O--(C1-C6)alkyl), or
--O--P(═O)(--O--(C1-C6)alkyl)2. In one embodiment
where R7 is --O--P(═O)(--O--(C1-C6)alkyl)2, the alkyl
groups are independently selected. In another preferred embodiment, R4 is
--CH2R7, where R7 is a carboxylic acid group or an amino carboxylic
acid group. In still another preferred embodiment R7 is a peptide; where
in a more preferred embodiment the peptide is linked through an ester
bond formed with the carboxyl terminal COOH group of the peptide chain to
the remainder of the compound. In other preferred separate and
independent embodiments of compounds of formula III, IIIa, IVa, IVb, Va,
or Vb where R4 is --CH2R7 and R7 is a peptide, the peptide may be a
dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Preferred
amino acid compositions for peptides of the R7 functionality are
described above.

[0162]Embodiments of compounds of formula III, IIIa, IVa, IVb, Va, or Vb
include those where X is selected from the group consisting of --(NR8)-,
S, and O, where R8 is independently selected from the group consisting of
hydrogen, --(C1-C6) alkyl, --(C1-C6) substituted
alkyl, --(C3-C9) cycloalkyl, --(C3-C9) substituted
cycloalkyl, and --O--(C1-C6) alkyl. Other embodiments of
compounds of formula III, IIIa, IVa, IVb, Va, or Vb include those where X
is --CH2--. In other embodiments of compounds of formula III, IIIa,
IVa, IVb, Va, or Vb, X is oxygen (O). In other embodiments of compounds
of formula III, IIIa, IVa, IVb, Va, or Vb, X is sulfur (S). In still
other embodiments of compounds of formula III, IIIa, IVa, IVb, Va, or Vb,
X is --(NR8)-, where R8 is independently selected from the group
consisting of hydrogen, --(C1-C6) alkyl, --(C1-C6)
substituted alkyl, --(C3-C9) cycloalkyl, --(C3-C9)
substituted cycloalkyl, and --O--(C1-C6) alkyl.

[0163]Other preferred embodiments of the invention include compounds of
formula III or Ma, where Q is a heteroaryl or an optionally substituted
heteroaryl group. In four separate alternative preferred embodiments of
compounds of formula III or IIIa, Q is an optionally substituted
monocyclic heteroaryl group, an optionally substituted bicyclic
heteroaryl group, an optionally substituted bicyclic heteroaryl group
with the proviso that the bicyclic heteroaryl group is not an indolyl
group or a substituted indolyl, or an optionally substituted tricyclic
heteroaryl group. Optional substituents, when present, are independently
selected from those recited for aryl, heteroaryl, --O-aryl, --S-aryl,
--O-heteroaryl, and --S-heteroaryl.

[0164]Included in the preferred embodiments of the invention are compounds
of formula IVa, IVb, Va, or Vb, where Q is a heteroaryl or an optionally
substituted heteroaryl group. In four separate alternative preferred
embodiments of compounds of formula IVa, IVb, Va, or Vb, Q is an
optionally substituted monocyclic heteroaryl group, an optionally
substituted bicyclic heteroaryl group, an optionally substituted bicyclic
heteroaryl group with the proviso that the bicyclic heteroaryl group is
not indolyl, or an optionally substituted tricyclic heteroaryl group. In
a more preferred embodiment, Q is an optionally substituted
nitrogen-containing heteroaryl group. In a related embodiment, Q is an
optionally substituted indolyl. Optional substituents, when present are
independently selected from those recited for aryl, heteroaryl, --O-aryl,
--S-aryl, --O-heteroaryl, and --S-heteroaryl.

[0165]Preferred embodiments of the invention include mixtures of compounds
of formula IVa and IVb, including racemic mixtures. In another preferred
embodiment, the compounds of formula IVa and IVb are the separate
enantiomers of
(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol--
3-yl)pyrrolidine-2,5-dione. In this embodiment the preparation of
(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol--
3-yl)pyrrolidine-2,5-dione is prepared as a mixture beginning with the
starting materials 1,2,3,4-tetrahydroquinoline and indole-3-acetamide.
The 1,2,3,4-tetrahydroquinoline is converted into
5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl) oxoacetic acid methyl
ester as described in Example 1, steps 1-5. The
5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl) oxoacetic acid methyl
ester is reacted with indole-3-acetamide as described in Example 1, step
6, to yield
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrol-
e-2,5-dione. The mixture of
(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol--
3-yl)pyrrolidine-2,5-dione is then prepared by catalytic hydrogenation as
described in Example 2 using Procedure B.

[0166]Preferred embodiments of the invention also include mixtures of
compounds of formula Va and Vb, including racemic mixtures. In another
preferred embodiment, the compounds of Va and Vb are the separate
enantiomers of
(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indo-
l-3-yl)pyrrolidine-2,5-dione. In this embodiment, the compounds are
prepared as a mixture by first preparing
(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol--
3-yl)pyrrolidine-2,5-dione, as described above. The mixture of cis
compounds is then treated with a mixture of potassium tert-butoxide in
tert-butanol to obtain a mixture of
(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indo-
l-3-yl)pyrrolidine-2,5-dione as described in Example 3.

[0167]All stereoisomers of the compounds of the instant invention are
contemplated, either in admixture or in pure or substantially pure form,
including crystalline forms of racemic mixtures and crystalline forms of
individual isomers. The definition of the compounds according to the
invention embraces all possible stereoisomers (e.g., the R and S
configurations for each asymmetric center) and their mixtures. It very
particularly embraces the racemic forms and the isolated optical isomers
having a specified activity. The racemic forms can be resolved by
physical methods, such as, for example, fractional crystallization,
separation or crystallization of diastereomeric derivatives, separation
by chiral column chromatography or supercritical fluid chromatography.
The individual optical isomers can be obtained from the racemates by
conventional methods, such as, for example, salt formation with an
optically active acid followed by crystallization. Furthermore, all
geometric isomers, such as E- and Z-configurations at a double bond, are
within the scope of the invention unless otherwise stated. Certain
compounds of this invention may exist in tautomeric forms. All such
tautomeric forms of the compounds are considered to be within the scope
of this invention unless otherwise stated. The present invention also
includes one or more regioisomeric mixtures of an analog or derivative.

[0169]As used herein, the term "metabolite" means a product of metabolism
of a compound of formula III, IIIa, IVa, IVb, Va, or Vb, or a
pharmaceutically acceptable salt, analog or derivative thereof, that
exhibits a similar activity in vivo to said a compound of formula III,
IIIa, IVa, IVb, Va, or Vb.

[0170]As used herein, the term "prodrug" means a compound of formula III,
IIIa, IVa, IVb, Va, or Vb covalently linked to one or more pro-moieties,
such as an amino acid moiety or other water solubilizing moiety. A
compound of formula III, IIIa, IVa, IVb, Va, or Vb may be released from
the pro-moiety via hydrolytic, oxidative, and/or enzymatic release
mechanisms. In an embodiment, a prodrug composition of the present
invention exhibits the added benefit of increased aqueous solubility,
improved stability, and improved pharmacokinetic profiles. The pro-moiety
may be selected to obtain desired prodrug characteristics. For example,
the pro-moiety, e.g., an amino acid moiety or other water solubilizing
moiety such as phosphate within R4, may be selected based on solubility,
stability, bioavailability, and/or in vivo delivery or uptake.

3. The Synthesis of Pyrroloquinolinyl-pyrrole-2,5-diones and
pyrroloquinolinyl-pyrrolidine-2,5-diones

[0171]Standard synthetic methods and procedures for the preparation of
organic molecules and functional group transformations and manipulations
including the use of protective groups can be obtained from the relevant
scientific literature or from standard reference textbooks in the field.
Although not limited to any one or several sources, recognized reference
textbooks of organic synthesis include: Smith, M. B.; March, J. March's
Advanced Organic Chemistry: Reactions, Mechanisms, and Structure,
5th ed.; John Wiley & Sons: New York, 2001; and Greene, T. W.; Wuts,
P. G. M. Protective Groups in Organic Synthesis, 3rd; John Wiley &
Sons: New York, 1999. The following descriptions of synthetic methods are
designed to illustrate, but not limit, general procedures for the
preparation of the compounds of formula III, IIIa, IVa, IVb, Va, or Vb.

3.1 General Procedures for the Synthesis of
pyrroloquinolinyl-pyrrole-2,5-dione and
pyrroloquinolinyl-pyrrolidine-2,5-diones where R4 is hydrogen

[0172]The present invention provides for
pyrroloquinolinyl-pyrrole-2,5-dione compounds of formula III, IIIa, IVa,
IVb, Va, or Vb. The preparation of compounds of formula III, IIIa, IVa,
IVb, Va, and Vb may be achieved by a series of reactions commencing with
the reaction of a 5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)
oxoacetic acid ester of formula I with an amide of formula II, to form a
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrol-
e-2,5-dione of formula III, including compounds of formula Ma, where R4 is
hydrogen, as shown in Scheme 1.

##STR00003##

3.1.1. Synthesis of
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrol-
e-2,5-diones of formula III where R4 is hydrogen

[0173]The condensation of an ester of formula I and a compound of formula
II to produce compounds of formula III, including compounds of formula
Ma, where R4 is hydrogen is conducted in any suitable anhydrous polar
aprotic solvent including, but not limited to, tetrahydrofuran (THF),
tetrahydropyran, diethyl ether and the like in the presence of base. For
the purposes of the reaction, suitable esters of formula I include, but
are not limited to, alkyl esters where R9 is a (C1-C4) alkyl group, and
preferred esters include the methyl and ethyl esters. Suitable bases for
the reaction include alkaline metal salts of low molecular weight alkyl
alcohols, including, but not limited to, alkaline metal salts of
methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, and
tert-butanol. Preferred alkaline metal salts of low molecular weight
alkyl alcohols include sodium and potassium salts, with potassium
tert-butoxide (tBuOK) being the preferred base. Typically the reactions
are conducted at 0° C. for 2 hours, however, both the time and
temperature may be altered depending upon the specific substituents
present on compounds of formula I and II, and the solvent employed. The
reaction temperature may be varied from -78° C. to 37° C.,
and is preferably from -35° C. to 25° C., or more
preferably from -15° C. to 10° C. Reaction times will
generally vary inversely with the temperature employed, suitable times
from about 15 minutes to 24 hours may be employed, more preferably, 30
minutes to 12 hours, and more preferably 1 to 6 hours.

3.1.2. Preparation of Compounds of Formula IVa, IVb, Va and Vb where R4 is
hydrogen

[0174]Reduction of compounds of formula III and Ma, where R4 is hydrogen
to yield the corresponding
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrol-
idine-2,5-diones having formula IVa, IVb, Va, or Vb may be conducted
employing a variety of procedures including, but not limited to,
reduction with zinc-mercury (Procedure A), catalytic hydrogenation
(Procedure B), and reduction with magnesium in methanol (Procedure C). As
indicated in Scheme 1, depending on the reduction reaction and conditions
chosen, the reaction will yield principally compounds of formula IVa and
IVb, or principally compounds of formula Va and Vb, or alternatively a
mixture of compounds of formula IVa, IVb, Va, and Vb.

[0175]Mixtures of compounds of formula IVa, IVb, Va, and Vb may be
prepared by the direct reduction of compounds of formula III or IIIa with
a zinc-mercury reducing agent. The reaction is generally carried out with
fresh reducing agent prepared by mixing Zn powder with HgCl2
deionized water followed by acidification with HCl. After drying, the
solid reducing agent (zinc-mercury) is suitable for reduction of
compounds of formula III or Ma in refluxing dry ethanol under a dry HCl
gas atmosphere as described in Example 2, Procedure A, for the reduction
of 3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyr-
role-2,5-dione.

[0176]An alternative method of preparing pyrrolidine-2,5-diones is
catalytic hydrogenation, which yields a mixture consisting principally of
the (±)-cis pyrrolidine-2,5-diones of formula IVa and IVb. Catalytic
hydrogenation of compounds of formula III or IIIa may be conducted in an
anhydrous alcohol over a noble metal catalyst under 1 atmosphere of
hydrogen for 48 hours. A variety of low molecular weight alkyl alcohols
may be employed to conduct the reduction, including n-propyl alcohol,
isopropyl alcohol, ethanol or methanol. Preferably the alcohol is ethanol
or methanol, and most preferably methanol. A noble metal catalyst (e.g.,
platinum, palladium, rhodium, ruthenium etc.) on charcoal is preferred
for the reduction of compounds of formula III or IIIa. In more preferred
embodiments, the noble metal catalyst is palladium on activated charcoal.
While reduction compounds of formula IIIa or III under 1 atmosphere of
hydrogen at room temperature (25° C.) for 12-48 hours is generally
suitable for preparation of pyrrolidine-2,5-diones, the pressure of
hydrogen, reaction time, and the reaction temperature may be varied.
Catalytic hydrogenation of
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrol-
e-2,5-dione is described in Example 2, Procedure B.

[0177]Pyrrole-2,5-diones of formula Ma or III may be reduced to yield a
mixture of compounds of formula Va and Vb by the reduction in anhydrous
alcohol with a metal reducing agent. Preferred metals include sodium,
calcium and magnesium, with magnesium as a more preferred metal reducing
agent. The reaction is typically carried out under an inert atmosphere of
nitrogen for 30 minutes to 2 hours by refluxing a compound of formula III
or formula IIIa in an alcohol selected from the group consisting of
methanol, ethanol, n-propanol, and isopropanol with magnesium turnings.
In preferred embodiments the reaction is conducted for about 40 minutes
in methanol as described in Example 2, Procedure C, for the preparation
of (±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-i-
ndol-3-yl)pyrrolidine-2,5-dione.

[0178]Compounds of IVa and/or IVb, which have the pyrrolidine ring
substituents in the cis configuration, may be converted into a mixture of
compounds of Va and Vb, where the substituents are in the trans
configuration, or into a mixture of all four isomers of formula IVa, IVb,
Va, and Vb by treatment with base in a polar protic solvent. Typically
the reaction employs an alkaline metal salt of a (C1-C4) alkyl alcohol in
an alcohol solvent (e.g., sodium or potassium methoxide in methanol,
sodium or potassium ethoxide in ethanol, sodium or potassium
tert-butoxide in tert-butanol), with potassium tert-butoxide in
tert-butanol as the preferred alkaline metal salt and solvent mixture.
Reactions are generally conducted from 0° C. to the reflux
temperature of the reaction mixture for 4 to 48 hours. In more preferred
embodiments, the reaction are conducted from room temperature (25°
C.) to the reflux temperature of the mixture for 8 to 24 hours, and in an
even more preferred embodiment, the reaction is conducted at about
50° C. in a mixture of potassium tert-butoxide in tert-butanol for
about 16 hours. Short reaction times and low temperatures favor formation
of mixtures still containing compounds IVa and/or IVb.

[0179]The introduction of additional substituted and unsubstituted aryl or
heteroaryl substituents on to aromatic rings of compounds of formula III,
IIIa, IVa, IVb, Va, or Vb may be accomplished by the reaction of a
substituted or unsubstituted aryl or heteroaryl boronic acid with an
aromatic halogen substituent on a compound of formula III, IIIa, IVa,
IVb, Va, or Vb. The reaction is typically carried out by heating a
mixture of a compound of formula III, IIIa, IVa, IVb, Va, or Vb bearing
an aryl or heteroaryl bromide or iodide, more preferably an arylbromide
or hetroarylbromide, with an aryl or heteroaryl boronic acid in the
presence of tetrakistriphenylphosphine palladium in a solvent mixture
consisting of 5 parts toluene, 5 parts ethanol, 1 part saturated
NaHCO3, and 2 parts water to 100° C. under nitrogen for 5
hours. After cooling to room temperature, the mixture is extracted with
ethyl acetate and concentrated. The residue is purified by silica gel
chromatography. In a preferred embodiment, the halogenated compound of
formula III, IIIa, IVa, IVb, Va, or Vb bears the halogen on an aryl or
heteroaryl group Q functionality resulting in the introduction of
substituted aryl or heteroaryl group donated by the boronic acid on to
the Q substituent. In a more preferred embodiment, the Q functionality is
a brominated aromatic or heteroaromatic Q functionality. In another more
preferred embodiment the halogenated Q functionality reacted with the
boronic acid is a halogenated 3-indolyl. Examples 31-34 describe the
introduction of substituted and unsubstituted aromatic groups into
compounds of formula Va and Vb employing a brominated Q functionality
where Q is a brominated 3-indolyl.

[0180]Aromatic and heteroaromatic boronic acids including 2-thienylboronic
acid, 3-thienylboronic acid, and 2-naphthylboronic acid are available
from a variety of commercial sources including Sigma-Aldrich (St. Louis,
Mo.). Alternatively aromatic and heteroaromatic boronic acids may be
prepared from the corresponding aryl or heteroaryl bromides by reaction
with triisopropyl borate in the presence of n-butyllithium followed by
quenching with aqueous HCl. (See, e.g., W. Li, et. al., J. Organic Chem.
67: 5394-97 (2002) and C. M. Marson, et. al., Tetrahedron 59: 4377-81
(2003).

[0181]Compounds of formula III, IIIa, IVa, IVb, Va, or Vb, where R4 is
hydrogen, can be converted into compounds of formula III, IIIa, IVa, IVb,
Va, or Vb where R4 is --CH2R7. The conversion begins with the
preparation of the hydroxymethylene derivative of the compounds as
indicated in the partial structures shown in Scheme 2.

##STR00004##

[0182]Preparation of the hydroxymethylene derivatives is accomplished by
reaction of a compound of formula III, IIIa, IVa, IVb, Va, or Vb where R4
is H with aqueous formaldehyde in tetrahydrofuran (THF). Typical reaction
conditions employ equal volumes of THF and 37% formaldehyde in water with
the reaction stirred for 14-16 hours at room temperature. Reaction times
and temperatures may vary from 1 hour to 48 hours and the temperature may
be from 0° C. to 50° C. or more preferably from 10°
C. to 37° C. Upon completion the reaction is partitioned between
water and an organic solvent, typically ethyl acetate. The organic layer
is dried over sodium sulfate, concentrated, and subject to chromatography
on silica gel as necessary to yield the hydroxymethylene product. The
preparation of the hydroxymethylene derivative of
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrol-
idine-2,5-dione,
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-1-hydroxymethyl-4-(1H-i-
ndol-3-yl)-pyrrolidine-2,5-dione, is described in Example 56, step 1.

[0183]Compounds of formula III, IIIa, IVa, IVb, Va, or Vb where R4 is
--CH2R7 and R7 is phosphate (--O--P(═O)(OH)2), monoalkyl
phosphate (e.g., --O--P(═O)(--OH)(--O--(C1-C6) alkyl)),
dialkyl phosphate (e.g., --O--P(═O)(--O--(C1-C6)
alkyl)2) a monobenzylphosphate ester (--O--P(═O)(--OH)
(--O--(CH2)-phenyl)), or a dibenzylphosphate ester
(--O--P(═O)(--O--(CH2)-phenyl)2) may be prepared from the
desired hydroxymethylene derivative and a suitably substituted phosphoric
acid by any reaction suitable for the formation of a phosphate ester bond
between the phosphoric acid compound and the hydroxymethylene derivative.
In a preferred method, the formation of phosphate esters is conducted by
reaction of a hydroxymethylene derivative of a compound of formula III,
IIIa, IVa, IVb, Va, or Vb with a suitably protected phosphoramidate
followed by deprotection. Reactions with the desired phosphoramidate are
typically conducted at room temperature in anhydrous THF. Following the
addition of the phosphoramidate, the reaction is treated with tetrazole
(3% in acetonitrile) and stirred 5 minutes to 1 hour, after which the
reaction is cooled to -78° C. The cooled reaction is treated with
m-chloroperbenzoic acid, and after stirring at -78° C. for 5
minutes, the reaction is warmed to room temperature and stirred for 5
minutes further. Following the removal of solvent, the product is
purified by flash chromatography on silica gel using ethyl acetate
hexane. The protecting groups are removed by suitable deprotection
reactions. Where the phosphoramidate employed is dibenzylphosphoramidate,
the benzyl protecting groups may be removed by hydrogenation of the
compound over Pd/C under 1 atmosphere of hydrogen at room temperature.
The preparation of phosphoric acid
mono-[3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-
-2,5-dioxo-pyrrolidin-1-ylmethyl]ester from
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-1-hydroxymethyl-4-(1H-i-
ndol-3-yl)-pyrrolidine-2,5-dione is described in Example 56, steps 2-3.

[0184]Compounds of formula III, IIIa, IVa, IVb, Va, or Vb where R7 is a
carboxylic acid group, or an amino carboxylic acid group, may be prepared
by coupling the desired hydroxymethylene derivative with a carboxylic
acid or amino carboxylic acid (amino acid) under conditions suitable for
the formation of an ester linkage. A variety of dehydrating agents,
including DCC (dicyclohexylcarbodiimide), HBTU
(0-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate),
or BOP ((benzotriazol-1-yloxy)tris(dimethylamino) phosphonium
hexafluorophosphate) may be employed to drive the formation of the ester
bond. In a preferred embodiment, the reactions are conducted in anhydrous
THF in the presence of HBTU and DIEPA (N,N-diisopropylethylamine) at room
temperature for 10 hours to 24 hours. Following completion of the
dehydration reaction, solvent is removed under reduced pressure and the
compounds are taken up in an organic solvent (e.g., ethyl acetate) and
washed with water. The organic layer is dried and the residue purified by
silica gel chromatography as necessary.

[0185]Where R7 is an amino carboxylic acid group, the starting materials
for introducing the amino carboxylic acid group must contain a suitably
protected amine A variety of suitable amine-protecting groups may be
advantageously employed including carbobenzyloxy-protected amines (e.g.,
the reactions may employ N-carbobenzyloxy glycine or N-carbobenzyloxy
alanine etc.). Subsequent deprotection will yield the free product. Where
the protecting group employed is carbobenzyloxy, deprotection may be
accomplished by treating the amine protected product suspended in
methanol with HCl (4M) in ethyl acetate in the presence of palladium on
charcoal (Pd/C) under 1 atmosphere of hydrogen for 1-3 hours at room
temperature. Examples 58-60 describe the preparation of compounds where
R7 is a carboxylic acid group, or an amino carboxylic acid group.

[0186]Compounds of formula III, IIIa, IVa, IVb, Va, or Vb where R7 is a
peptide, may be prepared by coupling the desired hydroxymethylene
derivative with a peptide bearing a free carboxylic acid group to form an
ester linkage. Linking of a carboxyl functionality of a peptide and the
hydroxymethylene group in an ester linkage may be conducted employing a
suitably protected peptide, bearing for example, protected free amine
groups protected with conventional N-protecting groups. Conditions
suitable for the formation of an ester linkage, include those employing
dehydrating agents, such as those described for the preparation of
compounds of formula III, IIIa, IVa, IVb, Va, or Vb where R4 is
--CH2R7 and R7 is a carboxylic acid group, or an amino carboxylic
acid group.

[0187]Compounds of formula III, IIIa, IVa, IVb, Va, or Vb where R4 is a
--(C1-C6) alkyl may be prepared by reacting by reacting the
desired compound of formula III, IIIa, IVa, IVb, Va, or Vb where R4 is H
with a (C1-C6) alkyl halide, where the halide is preferably Cl,
Br or I, in the presence of a suitable base at room temperature. Suitable
bases include organic bases such as potassium tert-butoxide, sodium
methoxide, and inorganic bases such as KOH, NaOH and K2CO3.
Suitable solvents include polar aprotic solvents such as DMSO, THF,
dioxane or other ethers, or DMF. In an alternative embodiment, the
compound of formula III, IIIa, IVa, IVb, Va, or Vb where R4 is H is
reacted with an organic or inorganic base to yield the conjugate base of
the compound of formula III, IIIa, IVa, IVb, Va, or Vb, and the conjugate
base is then reacted with the alkylhalide. Where the alkyl group is
introduced into a compound of formula III or IIIa, the resulting
alkylated compounds can be reduced to yield compounds of formula IVa and
IVb, Va and Vb, or a mixture of compounds of formula IVa, IVb, Va, and Vb
employing the reduction procedures described in Section I(b)(1). Example
61 describes the preparation of
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1-methylindol-3-yl)--
1-methylpyrrole-2,5-dione using iodomethane as an alkylating agent, and
its reduction by catalytic hydrogenation to yield
(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1-methyli-
ndol-3-yl)-1-methylpyrrolidine-2,5-dione.

[0188]Compounds of formula III, IIIa, IVa, IVb, Va, or Vb where R4 is a
--(C1-C6) alkyl group may also be prepared by reacting the
desired compound of formula III, IIIa, IVa, IVb, Va, or Vb where R4 is H
with a (C1-C6) alkyl alcohol in the presence of
diethylzodicarboxylate (DEAD) and triphenylphosphine. (See, e.g.,
Mitsunobu, O.; Wade, M.; Sano, T. J. Am Chem. Soc. 94: 694 (1972);
Hughes, D. L., Organic Reactions, 42; 335-656 (1992)). The reactions may
be conducted in a variety of solvents including tetrahydrofuran (THF),
dichloromethane, chloroform, acetonitrile, and benzene, preferably the
solvent is THF.

[0189]Where the isolation of an individual product having the structure of
formula III, IIIa, IVa, IVb, Va, or Vb is desired, the products may be
separated by chromatography on one or more chromatography media.
Chromatography may be carried out on a preparative scale or on an
analytical scale to determine the identity and purity of the products
present in a sample. Although any suitable chromatography media
including, but not limited to, silica, reverse phase, ion exchange,
chiral chromatographic media, or any combination thereof, may be
advantageously employed for separations, the suitability of specific
chromatographic media and conditions for the separation of products
having formula III, IIIa, IVa, IVb, Va, and Vb will depend upon the
substituents present on the compounds. In preferred embodiments,
chromatographic separations are conducted employing HPLC. In other
preferred embodiments the separation is carried out using supercritical
fluid chromatography. Where supercritical fluid chromatography is
employed, CO2, or mixtures of CO2 with other solvents including
acetonitrile (ACN), methanol, ethanol, isopropanol, or hexane, are the
preferred mobile phase, with mixtures of CO2 and methanol most
preferred. A variety of chromatographic media (stationary phases) may be
employed in supercritical fluid chromatography including, but not limited
to: ChiralCel OA, OB, OD, or OJ; ChiralPak AD or AS; Cyclobond I, II, or
III; and Chirobiotic T, V, and R media.

[0190]In more preferred embodiments, where the products are individual
isomers of formula IVa, IVb, Va, or Vb, mixtures containing two or more
of the isomeric forms may be separated by using supercritical fluid
chromatography on chiral media. In one more preferred embodiment,
separations are conducted on CHIRALPAK® AD columns (Daicel (U.S.A.)
Inc. Fort Lee, N.J.). In that embodiment, products are applied to the AD
column in a mixture of methanol and acetonitrile, or in acetonitrile, and
the column is subsequently eluted with 35% methanol in CO2 (65%). The
separation of
3(R),4(S)-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and
3(S),4(R)-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione on a CHIRALPAK® AD column is set forth in
Example 4. The separation of (+)
trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-
pyrrolidine-2,5-dione and (-)
trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-
pyrrolidine-2,5-dione is set forth in Example 5.

[0191]The individual racemic forms of compounds of formula III, IIIa, IVa,
IVb, Va, or Va may also be resolved by physical methods, such as, for
example, fractional crystallization or crystallization of diastereomeric
derivatives. In addition, individual optical isomers can be obtained from
racemic mixtures by conventional methods, such as, for example, salt
formation with an optically active acid, where applicable, followed by
crystallization.

3.2. Preparation of Compounds of Formula I and II where Y is a Bond

[0192]Compounds of formula I and II, which are employed in the synthesis
of pyrroloquinolinyl-pyrrole-2,5-dione of formula III and Ma, may be
purchased or obtained via a variety of synthetic routes such as those set
forth below.

3.2.1. Preparation of Compounds of Formula I where Y is a Bond

[0193]Compounds of formula I may be prepared from the corresponding
compound of formula A, where X is selected from the group consisting of
--(CH2)--, --(NR8)-, S and O, R8 is selected from the group
consisting of hydrogen, --(C1-C6) alkyl, --(C1-C6)
substituted alkyl, --(C3-C9) cycloalkyl, --(C3-C9)
substituted cycloalkyl, and --O--(C1-C6) alkyl, and m is 1 or
2. Exemplary compounds of formula A include 1,2,3,4-tetrahydroquinoline,
1,2,3,4-tetrahydro-quinoxaline, 3,4-dihydro-2H-benzo[1,4]oxazine,
3,4-dihydro-2H-benzo[1,4]thiazine, 2,3,4,5-tetrahydro-1H-benzo[b]azepine,
2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine,
6,7,8,9-tetrahydro-5-oxa-9-aza-benzocycloheptene, or
2,3,4,5-tetrahydro-benzo[b][1,4]thiazepine. The preparation begins with
the conversion of a compound of formula A to the corresponding
3-substituted-2-oxopropionic acid ethyl ester of formula B. The ethyl
ester of formula B is cyclized to form a compound of formula C, which is
converted to the free acid D, which is decarboxylated to yield the
desired tricyclic product E. Subsequent reaction of the tricyclic product
E with oxalyl chloride and work-up in alcoholic base yields the
corresponding compound of formula I. Scheme 3 illustrates the reaction
sequence beginning with compounds of formula A, which is further
illustrated in Example 1, steps 1-5 for the preparation of
5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl) oxoacetic methyl ester of
formula I from 1,2,3,4-tetrahydroquinoline and bromoethylpyrruvate
(3-bromo-pyruvic acid ethyl ester).

[0194]Some suitable conditions for the conversion of compounds of formula
A into compounds of formula I through the reaction sequence of Scheme 3
are described herein. Compounds of formula A may be converted to the
corresponding 3-substituted-2-oxopropionic acid ethyl ester of formula B
by treatment with bromoethyl pyruvate in an anhydrous ether, such as THF,
at room temperature for about 24 hours. Treatment of the
3-substituted-2-oxopropionic acid ethyl ester of formula B with anhydrous
MgCl2 in 2-methoxyethanol at about 125° C. for 30 minutes to
2 hours, preferably for 1 hour, results in the formation of the
corresponding tricyclic carboxylic acid ester of formula C. Subsequent
conversion of this compound to the free acid of formula D may be
accomplished by hydrolysis in aqueous base. In preferred embodiments the
reaction is carried out in an aqueous base, including but not limited to
NaOH or KOH, in the presence of alcohol as a co-solvent. Preferred
alcohol co-solvents include methanol, ethanol, n-propanol, and
isopropanol, with ethanol as a more preferred co-solvent. Reactions are
typically conducted by heating the mixture to reflux for 2 hours,
although the time and temperature of the reaction may be varied as
needed. Oxidative decarboxylation of compounds of formula D may be
conducted by a variety of procedures suitable for the decarboxylation of
aromatic acids. In preferred embodiments the decarboxylation of compounds
of formula D is conducted by heating the free acid with copper-chromite
(CuO--Cr2O3) in quinolone for about 2 hours to yield the
decarboxylated product of formula E. Conversion of compounds of formula E
to compounds of formula I may be accomplished by reaction with oxalyl
chloride, followed by treatment with a mixture of an anhydrous alcohol
and the alkaline metal salt of the alcohol, preferably sodium methoxide,
or sodium ethoxide. The reaction of oxalyl chloride with compounds of
formula E is typically conducted in anhydrous polar aprotic solvents
including ethers at a temperature from about -78° C. to about
10° C. In preferred embodiments, the reaction is conducted at a
temperature from about -25° C. to about 5° C. employing an
ether as a solvent. In more preferred embodiments the reaction is
conducted at 0° C. Preferred solvents for conducting the reaction
include, but are not limited to tetrahydrofuran (THF), tetrahydropyran,
diethyl ether and the like.

##STR00005##

3.2.2. Preparation of Compounds of Formula II

[0195]Compounds of formula II, which are substituted acetamides, may be
purchased or prepared from commercially available starting materials.
Commercially available acetamides including: indole-3-acetamide,
2-(5-methyl-1H-indol-3-yl)acetamide,
2-(5-methoxy-1H-indol-3-yl)acetamide,
2-(4-hydroxy-1H-indol-3-yl)acetamide, 2-phenylacetamide,
2-(4-methylphenyl)acetamide, 4-hydroxyphenylacetamide,
4-hydroxyphenylacetamide,
N-cyclopentyl-2-(4-hydroxy-2-oxo-1,2-dihydro-3-quinolinyl)acetamide,
2-phenoxyacetamide, 2-(2-methylphenoxy)acetamide,
2-(4-fluorophenoxy)acetamide, 2-(4-pyridinyl)acetamide, and
2-[(4-chlorophenyl)sulfanyl]acetamide are available from a variety of
sources including Sigma Aldrich Chemical Co., St. Louis Mo. A compound of
formula II may also be prepared from its corresponding free acid by
conversion of the free acid to its acid chloride followed by reaction
with ammonia.

3.3. Additional Routes for the Preparation of
Pyrroloquinolinyl-pyrrolidine-2,5-diones

[0196]In addition to those routes for the preparation of
pyrroloquinolinyl-pyrrolidine-2,5-diones described above, additional
routes of preparing the compounds exemplified for
(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indo-
l-3-yl)pyrrolidine-2,5-dione are described in Examples 62-64.

4. The Pharmaceutical Compositions and Formulations

[0197]A pharmaceutical composition of the invention is formulated to be
compatible with its intended route of administration. Examples of routes
of administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral (e.g., inhalation), transdermal (topical), and
transmucosal administration. Solutions or suspensions used for
parenteral, intradermal, or subcutaneous application can include the
following components: a sterile diluent such as water for injection,
saline solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as benzyl
alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic acid;
buffers such as acetates, citrates or phosphates, and agents for the
adjustment of tonicity such as sodium chloride or dextrose. The pH can be
adjusted with acids or bases, such as hydrochloric acid or sodium
hydroxide. The parenteral preparation can be enclosed in ampoules,
disposable syringes or multiple dose vials made of glass or plastic

[0198]A compound or pharmaceutical composition of the invention can be
administered to a subject in many of the well-known methods currently
used for chemotherapeutic treatment. For example, for treatment of
cancers, a compound of the invention may be injected directly into
tumors, injected into the blood stream or body cavities or taken orally
or applied through the skin with patches. The dose chosen should be
sufficient to constitute effective treatment but not so high as to cause
unacceptable side effects. The state of the disease condition (e.g.,
cancer, precancer, and the like) and the health of the patient should
preferably be closely monitored during and for a reasonable period after
treatment.

[0199]The term "therapeutically effective amount," as used herein, refers
to an amount of a pharmaceutical agent to treat, ameliorate, or prevent
an identified disease or condition, or to exhibit a detectable
therapeutic or inhibitory effect. The effect can be detected by any assay
method known in the art. The precise effective amount for a subject will
depend upon the subject's body weight, size, and health; the nature and
extent of the condition; and the therapeutic or combination of
therapeutics selected for administration. Therapeutically effective
amounts for a given situation can be determined by routine
experimentation that is within the skill and judgment of the clinician.
In a preferred aspect, the disease or condition to be treated is cancer.
In another aspect, the disease or condition to be treated is a cell
proliferative disorder.

[0200]For any compound, the therapeutically effective amount can be
estimated initially either in cell culture assays, e.g., of neoplastic
cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs.
The animal model may also be used to determine the appropriate
concentration range and route of administration. Such information can
then be used to determine useful doses and routes for administration in
humans. Therapeutic/prophylactic efficacy and toxicity may be determined
by standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., ED50 (the dose therapeutically effective in 50% of
the population) and LD50 (the dose lethal to 50% of the population).
The dose ratio between toxic and therapeutic effects is the therapeutic
index, and it can be expressed as the ratio, LD50/ED50.
Pharmaceutical compositions that exhibit large therapeutic indices are
preferred. The dosage may vary within this range depending upon the
dosage form employed, sensitivity of the patient, and the route of
administration.

[0201]Dosage and administration are adjusted to provide sufficient levels
of the active agent(s) or to maintain the desired effect. Factors which
may be taken into account include the severity of the disease state,
general health of the subject, age, weight, and gender of the subject,
diet, time and frequency of administration, drug combination(s), reaction
sensitivities, and tolerance/response to therapy. Long-acting
pharmaceutical compositions may be administered every 3 to 4 days, every
week, or once every two weeks depending on half-life and clearance rate
of the particular formulation.

[0202]The pharmaceutical compositions containing active compounds of the
present invention may be manufactured in a manner that is generally
known, e.g., by means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping, or
lyophilizing processes. Pharmaceutical compositions may be formulated in
a conventional manner using one or more pharmaceutically acceptable
carriers comprising excipients and/or auxiliaries that facilitate
processing of the active compounds into preparations that can be used
pharmaceutically. Of course, the appropriate formulation is dependent
upon the route of administration chosen.

[0203]Pharmaceutical compositions suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile injectable
solutions or dispersion. For intravenous administration, suitable
carriers include physiological saline, bacteriostatic water, Cremophor
EL® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In
all cases, the composition must be sterile and should be fluid to the
extent that easy syringeability exists. It must be stable under the
conditions of manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi. The
carrier can be a solvent or dispersion medium containing, for example,
water, ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), and suitable mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required particle
size in the case of dispersion and by the use of surfactants. Prevention
of the action of microorganisms can be achieved by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars, polyalcohols
such as manitol, sorbitol, sodium chloride in the composition. Prolonged
absorption of the injectable compositions can be brought about by
including in the composition an agent which delays absorption, for
example, aluminum monostearate and gelatin.

[0204]Sterile injectable solutions can be prepared by incorporating the
active compound in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that contains a
basic dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation are vacuum drying
and freeze-drying that yields a powder of the active ingredient plus any
additional desired ingredient from a previously sterile-filtered solution
thereof.

[0205]Oral compositions generally include an inert diluent or an edible
pharmaceutically acceptable carrier. They can be enclosed in gelatin
capsules or compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with excipients
and used in the form of tablets, troches, or capsules. Oral compositions
can also be prepared using a fluid carrier for use as a mouthwash,
wherein the compound in the fluid carrier is applied orally and swished
and expectorated or swallowed. Pharmaceutically compatible binding
agents, and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an excipient such as starch or lactose, a disintegrating agent
such as alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring
agent such as peppermint, methyl salicylate, or orange flavoring.

[0206]For administration by inhalation, the compounds are delivered in the
form of an aerosol spray from pressured container or dispenser, which
contains a suitable propellant, e.g., a gas such as carbon dioxide, or a
nebulizer.

[0207]Systemic administration can also be by transmucosal or transdermal
means. For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the formulation.
Such penetrants are generally known in the art, and include, for example,
for transmucosal administration, detergents, bile salts, and fusidic acid
derivatives. Transmucosal administration can be accomplished through the
use of nasal sprays or suppositories. For transdermal administration, the
active compounds are formulated into ointments, salves, gels, or creams
as generally known in the art.

[0208]In one aspect, the active compounds are prepared with
pharmaceutically acceptable carriers that will protect the compound
against rapid elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl
acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters,
and polylactic acid. Methods for preparation of such formulations will be
apparent to those skilled in the art. The materials can also be obtained
commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal suspensions (including liposomes targeted to infected cells
with monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared according to
methods known to those skilled in the art, for example, as described in
U.S. Pat. No. 4,522,811.

[0209]It is especially advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject to be
treated; each unit containing a predetermined quantity of active compound
calculated to produce the desired therapeutic effect in association with
the required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved.

[0210]In therapeutic applications, the dosages of the pharmaceutical
compositions used in accordance with the invention vary depending on the
agent, the age, weight, and clinical condition of the recipient patient,
and the experience and judgment of the clinician or practitioner
administering the therapy, among other factors affecting the selected
dosage. Generally, the dose should be sufficient to result in slowing,
and preferably regressing, the growth of the tumors and also preferably
causing complete regression of the cancer. Dosages can range from about
0.01 mg/kg per day to about 3000 mg/kg per day. In preferred aspects,
dosages can range from about 1 mg/kg per day to about 1000 mg/kg per day.
In an aspect, the dose will be in the range of about 0.1 mg/day to about
50 g/day; about 0.1 mg/day to about 25 g/day; about 0.1 mg/day to about
10 g/day; about 0.1 mg to about 3 g/day; or about 0.1 mg to about 1
g/day, in single, divided, or continuous doses (which dose may be
adjusted for the patient's weight in kg, body surface area in m2,
and age in years). An effective amount of a pharmaceutical agent is that
which provides an objectively identifiable improvement as noted by the
clinician or other qualified observer. For example, regression of a tumor
in a patient may be measured with reference to the diameter of a tumor.
Decrease in the diameter of a tumor indicates regression. Regression is
also indicated by failure of tumors to reoccur after treatment has
stopped. As used herein, the term "dosage effective manner" refers to
amount of an active compound to produce the desired biological effect in
a subject or cell.

[0211]Preferably,
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione is administered at dosage of 360 mg, twice a
day, for a maximal daily dosage of 720 mg.
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione is optionally administered to subjects or
patients at an initial dosage of 10 mg twice daily for a maximal daily
dose of 20 mg, with dosage escalation to administration of 360 mg twice
daily for a maximal daily dosage of 720 mg. Preferred dosage forms of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione include, but are not limited to, caplets,
tablets, pills, and freeze-dried powder. For instance, a subject or
patient is administered one 360 mg caplet twice a day, or alternatively,
two 180 mg caplets, twice a day, for a maximal daily dosage of 720 mg.
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione caplets or tablets are also formulated in 60 mg
doses.

[0212]The pharmaceutical compositions can include co-formulations of any
of the compounds described herein.

[0213]The pharmaceutical compositions can be included in a container,
pack, or dispenser together with instructions for administration.

EXAMPLES

[0214]Examples are provided below to further illustrate different features
of the present invention. The examples also illustrate useful methodology
for practicing the invention. These examples do not limit the claimed
invention.

[0216]To a solution of 1,2,3,4-tetrahydroquinoline (100 mL) in anhydrous
tetrahydrofuran (300 mL), bromoethylpyrruvate (53 mL) was added dropwise
over 30 minutes. The mixture was stirred for 24 hours at room
temperature. The reaction mixture was filtered and the solid washed with
tetrahydrofuran (100 mL). The filtrate was evaporated to dryness to give
3-(3,4-dihydro-2H-quinolin-1-yl)-2-oxopropionic acid ethyl ester as a
brown oil 117 g.

[0228]The active zinc-mercury reducing agent for the reduction of
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrol-
e-2,5-dione was prepared from metallic zinc and HgCl2. Zinc powder
(2.5 g) and mercury (II) chloride (0.25 g) were suspended in de-ionized
water (3 mL) and stirred for 20 minutes. A few drops of concentrated
hydrochloric acid was then added and the mixture stirred for few minutes.
The solid was filtered off, washed with de-ionized water (50 mL), ethanol
(50 mL) and dried.

[0229]To a suspension of the Zn(Hg) reducing agent prepared as above in
dry ethanol (50 mL) was added
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrol-
e-2,5-dione (0.35 g, 95.4 μmol.). The mixture was heated to reflux for
30-60 minutes while dry hydrogen chloride gas was slowly passed through
the mixture. The mixture was then cooled, filtered, and evaporated to
dryness. A 5% potassium carbonate solution (150 mL) and ethyl acetate
(300 mL) were then added. The organic layer was dried over anhydrous
magnesium sulfate and evaporated to give ˜2:1 mixture of
(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol--
3-yl)pyrrolidine-2,5-dione and
(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indo-
l-3-yl)pyrrolidine-2,5-dione (0.2 g).

Procedure B: Reduction of
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrol-
e-2,5-dione with hydrogen in the presence of palladium on carbon

[0239]A mixture of
(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indo-
l-3-yl)pyrrolidine-2,5-dione (200 mg) in acetonitrile (1 mL) was subjected
to preparative supercritical fluid chromatography using a CHIRALPAK®
AD column (Daicel, U.S.A.) 20 mm×250 mm, eluting with 35%
methanol/CO2 at a flow rate of 3.5 mL/minutes. Chromatography yielded a
faster eluting peak of the trans isomer (82 mg) having a negative optical
rotation assigned
(-)-3(R),4(R)-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-ind-
ol-3-yl)pyrrolidine-2,5-dione and a slower eluting peak of the trans
isomer (86 mg) having a positive optical rotation assigned
(+)-3(S),4(R)-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-ind-
ol-3-yl)pyrrolidine-2,5-dione. Absolute stereochemical assignments were
based solely upon relative retention time of related compounds they may
be reversed. All optical rotation measurements were conducted in
chloroform at 25° C. at 589 nm.

[0240]Crystals of the chromatographically separated (+) or (-) isomers of
trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-
pyrrolidine-2,5-dione may be prepared from 2,2,2-trifluoroethanol using
vapor stress techniques and slow evaporation at 49° C. Crystals of
these isomers may also be prepared from ethanol at room temperature by
evaporation employing seed crystals, such as those prepared by vapor
stress techniques.

[0292](±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-
-indol-3-yl)pyrrolidine-2,5-dione (3.0 g, 8.13 mmol, prepared as in
Example 2, Procedure C) and formaldehyde (30 mL, 37% in water) in
tetrahydrofuran (30 mL) were stirred for 14-16 hours at room temperature.
The mixture was then taken up in ethyl acetate (50 mL) and water (50 mL).
The organic layer was washed with brine and dried over sodium sulfate.
Solvent was removed under reduced pressure and residue was purified using
a silica gel chromatography column eluted with EtOAc/Hexane 1:1 to yield
2.5 g, 77%, of
(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-1-hydroxym-
ethyl-4-(1H-indol-3-yl)-pyrrolidine-2,5-dione an orange foamy solid (2.5
g, 77%).

[0298]To a solution of
(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-1-hydroxym-
ethyl-4-(1H-indol-3-yl)-pyrrolidine-2,5-dione (0.5 mmol) in
tetrahydrofuran (8 mL) was added N-carbobenzyloxy alanine (1.1
equivalents) followed by the addition of HBTU (1.5 equivalents) and DIPEA
(2.2 equivalents). The mixture was stirred at room temperature for 15 h.
The solvents were removed under reduced pressure and the residue was
taken up in ethyl acetate and water (1:1, 15 mL). The organic layer was
separated and dried. The residue was purified by silica gel
chromatography to provide the N-carbobenzyloxy protected product.

[0306](±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-
-indol-3-yl)pyrrolidine-2,5-dione may be prepared by reacting 1H-indole
and 3,4-dibromo-1-phenyl-pyrrole-2,5-dione in the presence of methyl
magnesium bromide to yield
3-bromo-4-(1H-indol-3-yl)-1-phenyl-pyrrole-2,5-dione. The
3-bromo-4-(1H-indol-3-yl)-1-phenyl-pyrrole-2,5-dione is subsequently
reacted with 5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinoline and LiHMDS
(lithium hexamethyldisilane) in toluene or
(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-boranediol and
Pd(PPh3)4 (tetrakis(triphenylphosphine)palladium) to yield
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-1-phe-
nyl-pyrrole-2,5-dione, which is reduced and deprotected by treatment with
Mg in methanol, as in Example 2 procedure C, followed by catalytic
hydrogenation over palladium on carbon to yield
(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indo-
l-3-yl)pyrrolidine-2,5-dione. Bnz is benzyl.

[0307](±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-
-indol-3-yl)pyrrolidine-2,5-dione may be prepared by reacting
1-allyl-7-bromo-1H-indole with (COCl)2 (oxalyl chloride) and sodium
methoxide in a polar aprotic solvent such as dichloromethane to yield
(1-allyl-7-bromo-1H-indol-3-yl)-oxo-acetic acid methyl ester, which is
subsequently reacted with 2-(1H-indol-3-yl)-acetamide and tBuOK
(potassium tert-butoxide) in THF to yield
3-(1-allyl-7-bromo-1H-indol-3-yl)-4-(1H-indol-3-yl)-pyrrole-2,5-dione.
Reduction of the
3-(1-allyl-7-bromo-1H-indol-3-yl)-4-(1H-indol-3-yl)-pyrrole-2,5-dione by
Mg in refluxing methanol, as in Example 2 procedure C, yields
3-(1-allyl-7-bromo-1H-indol-3-yl)-4-(1H-indol-3-yl)-pyrrolidine-2,5-dione-
, which is treated with 9-BBN (9-borabicyclo[3.3.1]nonane) and
Pd(PPh3)4 (tetrakis(triphenylphosphine)palladium) to yield
(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indo-
l-3-yl)pyrrolidine-2,5-dione.

[0308]The cis and trans isomers of
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrol-
idine-2,5-dione may be prepared beginning with the reaction of
(1H-indol-3-yl)-oxo-acetic acid methyl ester and
(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-acetic acid methyl ester
in the presence of a base such as LDA (lithium diisopropylamide) in a
polar aprotic solvent such as THF to yield
2-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-3-(1H-indol-3-yl)-but-2-
-enedioic acid dimethyl ester. Alternatively,
2-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-3-(1H-indol-3-yl)-but-2-
-enedioic acid dimethyl ester may be prepared by reaction of
(1H-indol-3-yl)-acetic acid methyl ester and
(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-oxo-acetic acid methyl
ester in the presence of a base (e.g., LDA) in THF. The
2-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-3-(1H-indol-3-yl)-but-2-
-enedioic acid dimethyl ester is reduced by catalytic hydrogenation over a
noble metal catalyst (e.g., Pd on charcoal) to give
2-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-3-(1H-indol-3-yl)-succi-
nic acid dimethyl ester, which is reacted with benzylamine
(PhCH2NH2) to yield a mixture of cis and trans
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-1-phe-
nyl-pyrrolidine-2,5-dione. The mixture of cis and trans isomers may be
deprotected by catalytic hydrogenation over Pd on charcoal (Pd--C) to
give rise to a mixture of cis and trans
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-pyrro-
lidine-2,5-dione. The cis and trans isomers may be separated to give all
four cis and trans isomers (e.g., by chromatography as in Examples 4 and
5). The deprotected mixture of cis and trans isomers may be treated with
potassium tert-butoxide in tert-butanol (as in Example 3) or a mixture of
THF and tert-butanol at 50° C. to yield a mixture with a
predominance of the trans isomers. Alternatively, the
2-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-3-(1H-indol-3-yl)-succi-
nic acid dimethyl ester can be reacted with ammonia in methanol at
elevated temperatures to yield predominantly the cis isomers of
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-pyrro-
lidine-2,5-dione, which may be isomerized to yield predominately the trans
isomers of
3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-pyrro-
lidine-2,5-dione with potassium tert-butoxide in tert-butanol (as in
Example 3) or a mixture of THF and tert-butanol at 50° C.

[0335]Isobologram analysis: For each cell line and drug combination, the
72 hour IC50 values were determined for each individual drug and in
combination at the equipotent fixed ratio by MTT proliferation endpoint
assay or by colony formation assay. For example, in the case of drugs A
and B, where the IC50 values are 1 μM and 5 μM, respectively,
the equipotent ratio is 1:5. Therefore, a serial dilution of the highest
combination concentration (8× to 0.125X, where X is the IC50
ratio concentration) was used to generate a dose response curve. The
degree of inhibition of cell proliferation in this assay relative to
unexposed controls was designated the "effect", which ranged from 0.0 (no
inhibition) to 1.0 (no cellular conversion of the MTT or MTS reagent,
denoting complete cell death). Duplicate independent experiments were
performed for each cell line/drug combination. Data were then analyzed by
Calcusyn® (Biosoft, Cambridge, UK) data analysis software.

[0336]Cell survival analysis. In some experiments, cell survival was
determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay. Briefly, cells were plated in a 96-well plate at
5-10,000 cells per well, cultured for 24 hours in complete growth medium,
and then treated with various drugs and drug combinations for 72 hours.
MTT was added to a final concentration of 0.5 mg/mL, and incubated for 1
hour, followed by assessment of cell viability using the microplate
reader at 570 nm. Data were normalized to untreated controls and analyzed
with Microsoft Excel.

[0337]Cell proliferation assay. Exponentially growing cells were seeded at
2,000 cells per well in 6-well plates and allowed to attach for 24 hours.
Increasing concentrations of individual drugs and those in combination
were then added to the media for another 24 hours. After 24 hours
exposure, the drug was removed and fresh media was added for the next
14-21 days, allowing for colony formation. Cells were fixed and stained
with GIEMSA (Gibco BRL). Colonies of greater than 50 cells were scored as
survivors and the percentage of cell survival was plotted to determine
the IC50 values.

[0338]The studies described herein used a compound of Formula Va shown
herein, namely,
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione, a small molecule inhibitor of the c-Met
receptor tyrosine kinase, in combination with the multi-targeted kinase
inhibitor, sorafenib.

[0341]Combination indices were determined according to the method of Chou.
{Chou, T.-C. 1991. The median-effect principle and the combination index
for quantitation of synergism and antagonism, p. 61-102. In T.-C. Chou
and D. C. Rideout (ed.), Synergism and antagonism in chemotherapy.
Academic Press, San Diego, Calif}. FIG. 2, panel A, shows where
CAO and CBO are the separate concentrations of drug A
and B respectively that achieve the same effect as the mixture of drug A
and of drug B. Isobologram analyses categorized drug combinations as
synergistic, additive, or antagonistic. Specifically, FIG. 2, panel B,
shows isobologram analyses for a particular effect (e.g., 50% of the
maximum) in which the dose of drug A alone is A=20 and drug B alone is
B=100. The straight line connecting these intercept points is the
additivity line which, based on these potencies, should give the same
effect. An actual dose pair such as point Q attains this effect with
lesser quantities and is supra-additive or synergistic, while the dose
pair denoted by point R means greater quantities are required and is
therefore sub-additive. A point such as P that appears below the line
would be simply additive.

[0342]The combination data of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione with sorafenib are shown in Table 2 and the
combination data with sunitinib are shown in Table 3. The compound
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione is identified as "Agent A" in these tables. The
identity and tissue origin of cancer cell lines are indicated. The
results show that the combination
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and sorafenib revealed synergistic cytotoxicity
in 3 NSCLC cell lines (NCI-H522, NCI-H358, NCI-H460), MDA-MB-231
(breast), A375 (melanoma), the HCC1395 breast cancer line, the Caki-1
renal cell carcinoma, the HeLa cervical carcinoma cell line, and the A431
epidermoid carcinoma and showed additive cytotoxicity in 40 other cell
lines including, but not limited to, the Colo205 and SW480 colon cancer
lines, the NCI-H358 (NSCLC) cell line, and 3 hepatocellular carcinomas
(JHH-4, PLC/PRF/5, SK-Hep-1). Table 2 shows the combination cytoxicity of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and sorafenib in human cancer cell lines in
which either additivity or synergism (supra-additivity) were observed.
Table 3 shows the combination cytoxicity of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and sunitinib in human cancer cell lines in
which either additivity or synergism (supra-additivity) were observed.

[0343]The anti-proliferative effect of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and sorafenib on the NCI-H522 NSCLC cell line in
vitro was also assessed. Cells were treated with increasing
concentrations of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione from 0.14 to 33 μM and sorafenib from 0.015
to 100 μM for 72 hr. Cell growth was assessed using a standard
commercially available MTS reagent [3-(4,5-dimethylthiazol-2-yl)-5-(3
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt from
Promega, Madison, Wis.] assay. An isobologram was plotted and the
combination index was determined which showed the synergism of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and sorafenib.

[0344]The in vivo anti-proliferative effect of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and sorafenib was also assessed. Female Ncr
nu/nu mice with established subcutaneous NCI-H522 NSCLC tumors were
treated by oral gavage with the indicated doses of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione, sorafenib, both agents, or vehicle control for
21 days (days 8-29). All regimens were orally administered once daily for
21 days. Tumor sizes were evaluated periodically during treatment at the
indicated days post-inoculation. FIG. 3 is a graphical representation of
the combination treatment of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione (shown as Agent A) and sorafenib demonstrating
an effect in the NCI-H522 NSCLC xenograft model. These results are
represented as the mean of tumor weight±SEM of 10 tumors during the
treatment period. Both compounds were well-tolerated in all cohorts, and
no adverse effects on body weight gain were observed, showing that these
two agents are eminently combinable in vivo.

[0345]Taken together, these pre-clinical data demonstrate that the
combinational therapy of c-Met inhibitors, such as
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione, and kinase inhibitors, such as sorafenib, shows
highly encouraging anti-proliferative activity against a wide range of
cancer cell lines, in vitro and in vivo.

Example 77

Combination of c-Met Inhibitors and Sorafenib for the Treatment of
Microphthalmia Transcription Factor-associated Tumors

[0346]In clinical studies, monotherapy treatment with
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione has been well tolerated and has resulted in
tumor responses and prolonged stable disease across broad ranges of
tumors and doses. Indications with favorable clinical treatment include,
MiT (Microphthalmia Transcription Factor)-associated tumors, non-small
cell lung cancer and pancreatic adenocarcinoma. MiT tumors, which include
clear cell sarcoma (CCS), alveolar soft part sarcoma (ASPS) and
translocation-associated renal cell carcinoma (RCC), are linked
biologically through a common chromosomal abnormality that is responsible
for the over-expression of c-Met resulting in the development of these
tumors. Similar clinical studies are directed to hepatocellular carcinoma
(HCC) both for monotherapy treatment and combinational therapy with
kinases inhibitors, such as sorafenib.

[0347]According to the National Cancer Institute, 21,370 new cases of HCC
in the United States were projected in 2008, and 18,410 deaths were
projected to be caused by the disease. In the U.S., the increasing
incidence of HCC is related primarily to hepatitis C infection and
cirrhosis. The first drug to be approved for patients with unresectable
HCC was sorafenib. For patients who experience disease progression
following sorafenib treatment or for those patients unable to tolerate
sorafenib, no alternative therapy with proven clinical benefit is
available. Thus, there is a high unmet medical need for novel treatment
approaches in patients with advanced HCC for whom sorafenib treatment is
not an option.

[0348]Over-expression of c-Met and its ligand, hepatocyte growth factor
(HGF), is associated with poor prognoses in patients with HCC. When
abnormally activated, c-Met plays multiple roles in aspects of human
cancer, including cancer cell growth, survival, angiogenesis, invasion
and metastasis. Scientific literature related to HCC provides evidence of
the aberrant activation of the c-Met pathway. In addition, the
dysregulation of c-Met and HGF has been shown to be common in this
disease. Cell proliferation is a central mechanism responsible for liver
cancer progression, and c-Met is believed to play an important role in
this process.

[0349]In these clinical studies combining
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and sorafenib, Patients are treated with
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione at 360 mg twice daily (bid) and sorafenib at 200
mg bid. Patients are also screened to valuate dynamic changes of
hepatocyte growth factor (HGF), vascular endothelial growth factor
(VEGF), and soluble c-Met in patients' peripheral blood that are
associated with treatment of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and sorafenib. The results of the combinational
treatment show additive and synergistic anti-proliferative effects when
compared to monotherapy treatment with
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione.

Example 78

Combination of c-Met Inhibitors and Kinase Inhibitors for the Treatment of
Various Anti-Proliferative Disorders and Cancer

[0351]In vitro combination cytotoxicity studies were conducted with
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione, in combination with sorafenib and sunitinib
against a large panel of human cancer cell lines. A wide range of
combination effects was observed, with a higher incidence of either
synergistic or additive cytotoxic effects documented with
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and sorafenib as compared with
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and sunitinib. Interestingly, the two cell lines
where (-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-i-
ndol-3-yl)pyrrolidine-2,5-dione and sunitinib showed synergistic
cytotoxicity are both known to express activated c-Met due to c-Met gene
amplification (the SNU-16 gastric carcinoma and the SCH gastric
choriocarcinoma cell line) (Table 3). The compound
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione is identified as "Agent A" is this table. While
the effects observed were not tissue-type specific, the cell lines
exhibiting synergy with
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and sorafenib included two c-Met expressing
breast carcinoma cell lines (HCC1395 and MDA-MB-231) and three non-small
cell lung carcinomas (NSCLC), NCI-H460, NCI-H358 and NCI-H522, all with
documented c-Met expression or gene amplification. The NCI-H522 NSCLC
cell line is known to exhibit c-Met gene amplification and secrete high
levels of HGF, but it does not exhibit high constitutive levels of
phospho-c-Met upon serum starvation. Nonetheless,
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and sorafenib demonstrated augmented anti-tumor
efficacy when co-administered orally in an athymic mouse xenograft model
(FIG. 3). (-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(-
1H-indol-3-yl)pyrrolidine-2,5-dione and sorafenib showed additive
cytotoxicity in 5 hepatocellular carcinomas (HCC) tested, a clinical
indication for which sorafenib is currently approved. Recapitulation of
the synergistic or additive cytotoxic effects of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione and marketed TKIs in relevant animal models
guide potential clinical development strategies for
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione, a novel, orally administered and well-tolerated
anti-cancer drug candidate that demonstrates indications of clinical
activity.

[0352]The combination data of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5-dione (shown as Agent A in FIG. 4) with either
sorafenib or sunitinib were expressed as 1/Combination Index as shown in
FIGS. 4 and 5.

Example 79

Combination of c-Met Inhibitors and Erlotinib for the Treatment of
Non-Small Cell Lung and Colon Cancer

[0353]The effects of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with erlotinib were tested in NCI-H441
non-small cell lung cancer cells (NSCLC). Erlotinib is an inhibitor of
the epidermal growth factor receptor and is indicated for the treatment
of patients with locally advanced or metastatic non-small cell lung
cancer after failure of at least one prior chemotherapy regimen, and is
indicated for the first-line treatment of patients with locally advanced,
unresectable or metastatic pancreatic cancer. The IC50 of erlotinib
was predicted to be approximately 1 μM, while the IC50 of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione was predicted to be 300 nM for NCI-H441 NSCLC
cells and therefore an
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione:erlotinib ratio of 1:3 was used. A 1:33
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione:erlotinib was used for HT29 colon cancer cells.
The CI ranged between 0.45-1 at the ED50 for the NCI-H441 cell line
as determined by independent experiments, and the CI was 0.72 for the
Ht29 cell line (Table 4). The compound
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione is identified as "Agent A" in this table.
Median effect plots and isobolograms were performed for each experiment.
This data demonstrates an additive to synergistic anti-proliferative
effects of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione and erlotinib in both non-small cell lung
cancer cells (NSCLC) and colon cancer cells.

[0354]The effects of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with erlotinib were tested in a NCI-H441 NSCLC
human tumor xenograft model.
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione was administered daily at 300 mg/kg orally,
five days a week for four weeks (qd×5×4). Erlotinib was
administered daily at 100 mg/kg or 50 mg/kg orally, five days a week for
four weeks. As shown in Table 5 and FIG. 6, in all studies, the
combination of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with erlotinib showed improved
anti-proliferative effects over monotherapy treatment and the data
demonstrates at least an additive, and possibly synergistic, effect of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with erlotinib in non-small cell lung cancer.
The compound
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione is identified as "Agent A" Table 5 and FIG. 6.

Combination of c-Met Inhibitors and Gefitnib for the Treatment of Colon
Cancer and Lung Cancer

[0355]The effects of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with gefitinib were tested in HT29 colon cancer
cells. Gefitinib is an inhibitor of the EGF receptor. The IC50 of
gefitinib was predicted to be approximately 5 μM, while the IC50
of (-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indo-
l-3-yl)pyrrolidine-2,5,-dione was predicted to be 150 nM for HT29 colon
carcinoma cells, therefore an
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione:gefitinib ratio of 1:33 was used. The CI was
1.27 at the ED50 (Table 6). The compound
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione is identified as "Agent A" is this table.
Median effect plots and isobolograms were performed for each experiment.
This data demonstrates at least an additive anti-proliferative effect of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione and gefitinib in colon cancer cells.

[0356]The effects of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with gefitinib were tested in a NCI-H441 human
lung tumor xenograft model.
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione was administered daily at 300 mg/kg orally,
five days a week for four weeks (qd×5×4). Gefitinib was
administered daily at 50 mg/kg orally, five days a week for four weeks.
As shown in Table 7 and FIG. 7, in all studies, the combination of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with gefitinib showed improved
anti-proliferative effects over monotherapy treatment and the data
demonstrates at least an additive, and possibly synergistic, effect of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with gefitinib in non-small cell lung cancer.
The compound
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione is identified as "Agent A" Table 7 and FIG. 7.

Combination of c-Met Inhibitors and Carboplatin for the Treatment of
Pancreatic Cancer

[0357]The effects of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with the DNA synthesis inhibitor carboplatin,
was tested in MIA PaCa-2 pancreatic tumor cells. Carboplatin has been
reported to be beneficial in both pancreatic and prostate cancer when
combined with other therapeutic agents. The IC50 of carboplatin was
predicted to be approximately 25-50 μM, while the IC50 of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione was predicted to be 150 nM for MIA PaCa-2
cells. An (-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(-
1H-indol-3-yl)pyrrolidine-2,5,-dione:carboplatin ratio of 1:50 was used,
unless otherwise indicated, due to the insolubility of carboplatin at
higher concentrations. For MIA PaCa-2 cells the CI ranged between
1.09-1.45 at the 50% effective dose (ED50) (Table 8). The compound
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione is identified as "Agent A" is this table.
Median effect plots and isobolograms were performed for each experiment.
This data demonstrates at least an additive anti-proliferative effect of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with carboplatin in pancreatic cancer cells.
The mild variability observed in this drug combination is likely due to
solubility issues of carboplatin at the higher dose ranges.

Combination of c-Met Inhibitors and Cisplatin for the Treatment of
Pancreatic Cancer

[0358]The effects of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with the DNA synthesis inhibitor cisplatin were
tested in MIA PaCa-2 pancreatic tumor tumor cells. The IC50 of
cisplatin was predicted to be approximately 5-15 μM, while the
IC50 of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione was predicted to be 150 nM for MIA PaCa-2
cells. An (-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(-
1H-indol-3-yl)pyrrolidine-2,5,-dione:cisplatin ratio of 1:50 was used
unless otherwise indicated. For MIA PaCa-2 cells the CI ranged between
0.74-0.79 at the ED50 (Table 9). The compound
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione is identified as "Agent A" is this table.
Median effect plots and isobolograms were performed for each experiment.
This data demonstrates a synergistic anti-proliferative effect of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione and cisplatin in pancreatic cancer cells.

Combination of c-Met Inhibitors and Various Chemotheraputic Agents for the
Treatment of Gatric Cancer

[0359]The effects of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with 5-FU, TS-1, Capecitabine, and cisplatin
(CDDP) were tested in MKN-45 human gastric tumor xenograft model (FIG.
11-14). (-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-
-indol-3-yl)pyrrolidine-2,5,-dione was administered daily at 300 mg/kg
orally, five days a week for two weeks (qd×5×2). 5-FU was
administered daily at 10 mg/kg intravenously, five days a week for two
weeks. TS-1 was administered daily at 10 mg/kg orally, five days a week
for two weeks. Capecitabine was administered daily at 360 mg/kg orally,
five days a week for two weeks. CDDP was administered weekly at 5 mg/kg
intravenously for two weeks. As shown in Table 10, in all studies, the
combination of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with these compounds showed improved
anti-proliferative effects over monotherapy treatment and the data
demonstrates at least and additive anti-proliferative effect of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with 5-FU, TS-1, Capecitabine, and cisplatin in
gastric cancer.

Combination of c-Met Inhibitors and Imatinib for the Treatment of Colon
Cancer

[0360]The effects of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with imatinib (Gleevec) were tested in HT29
colon cancer cells. Imatinib is an inhibitor of the Abelson
proto-oncogene, c-kit, and PDGF-R (platelet-derived growth factor
receptor) and is indicated for the treatment of chronic myelogenous
leukemia (CML), gastrointestinal stromal tumors (GISTs) and a number of
other malignancies. The IC50 of imatinib was predicted to be
approximately 10 μM, while the IC50 of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione was predicted to be 150 nM for HT29 cells,
therefore an
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione:imatinib ratio of 1:66 was used. The CI was
1.22 at the ED50 (Table 11). The compound
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione is identified as "Agent A" is this table.
Median effect plots and isobolograms were performed for each experiment.
This data demonstrates a nearly additive anti-proliferative effect (a CI
of 1.22 nearly meeting the criterion of a CI=1.2) of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione and imatinib in colon cancer cells.

[0363]To potentially inform phase II clinical trials, in vitro combination
studies were performed using
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with gemcitabine, the current drug standard of
care used for pancreatic cancer treatment. The purpose of this study was
to independently recapitulate the effects of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione and gemcitabine combination on human pancreatic
cancer cell lines using a different method of calculation and comparing
with the data obtained by the CalcuSyn® software (Biosoft).

[0364]MIA PaCa-2 (also referred to as PACA2), PANC-1, CFPAC-1, and Hs766T
human pancreatic cell lines were maintained in DMEM supplemented with 10%
fetal bovine serum (FBS), penicillin, streptomycin, and fungizone. AsPC-1
cells were maintained in RPMI supplemented with 10% FBS, penicillin,
streptomycin, and fungizone. HPAF-II cells were maintained in MEM
supplemented with 10% FBS, penicillin, streptomycin, and fungizone. For
the MTS cytotoxicity assay, cells were plated in 96-well plates at 2,000
cells per well and incubated with increasing concentrations of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione and gemcitabine in combination for 72 hr. MTS
reagent (Promega, Madison, Wis.) was added to each well and plates were
incubated for 4 hr at 37° C. The absorbance of each well was
measured at 492 nm using a microplate reader. A preliminary experiment
with (-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-in-
dol-3-yl)pyrrolidine-2,5,-dione or gemcitabine (GEM) alone was performed
to determine the IC50 of each individual compound on each cell line
and a concentration range was also determined, The layout of dose range
of (-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indo-
l-3-yl)pyrrolidine-2,5,-dione (shown as Agent A in FIG. 8) and gemcitabine
for drug combination analysis was further determined. The IC50
calculation and IC50 value determinations are shown in Table 12 and
FIG. 8.

[0365]The results of these studies show that the combination of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione and gemcitabine provides a synergistic
anti-proliferative effect in PANC-1, HPAF-II and AsPC-1 human pancreatic
cancer cell lines. All three pancreatic cell lines were sensitive to the
combinatorial treatment of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione and gemcitabine. The results of this study
recapitulate previous work and confirm that the combination of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione and gemcitabine is synergistic in 3 out of the
6 pancreatic cell lines tested.

Combination of c-Met Inhibitors and Taxotere for the Treatment of
Pancreatic Cancer, Colon Cancer and Prostate Cancer

[0366]The effects of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with taxotere were tested in MIA PaCa-2
pancreatic tumor cells, PC-3 prostate tumor. For each cell line, the
IC50 value of taxotere was predicted to be approximately 5 nM, while
the IC50 of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione was predicted to be 150 nM for MIA PaCa-2
cells, and 1 μM for PC-3 cells. For these cell lines
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione:taxotere ratios of 200-1000:1 were used,
depending on predicted IC50 values. For MIA PaCa-2 cells the CI was
0.99 at the ED50, (Table 15) and therefore the effects of the
combination were additive. The compound
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione is identified as "Agent A" is this table.
Median effect plots and isobolograms were performed for each experiment.

[0367]This combination was also tested in PC-3 cells, where the
combination index ranged between 0.68 and 1.51 at ED50 (Table 16).
The compound
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione is identified as "Agent A" is this table.

[0368]It has been demonstrated that
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione and taxotere has a beneficial effect when dosed
in combination in xenograft studies. While the 72 hour MTT data suggested
that the combination of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione and taxotere was additive, the MTT assay may be
the most accurate assay of cell death due to taxotere's mechanism of
action of cell death. Therefore, additional combination cell death assays
using colony formation were performed in order to capture the long term
effects of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione and taxotere on cell death. Colony formation
assays were performed in PC-3, HT29, and MIA PaCa-2 cells to determine
whether the effects of this combination were synergistic. By colony
formation assay the CI for MIA PaCa-2 cells was 0.43, which indicates
synergism. Slight synergism to additivity was also observed for HT-29
cells, whereas the
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione:taxotere mixture was additive for PC-3 cells.

[0371]To potentially inform phase II clinical trials, an in vitro
combination study was initiated using
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with a number of chemotherapeutic agents
(gemcitabine, docetaxel and carboplatin) that are currently in clinical
use.

[0372]MIA PaCa-2 (also referred to as PACA2), PANC-1, CFPAC-1, Hs766T,
DU-145, PC-3 and SK-OV-3 cells were maintained in DMEM supplemented with
10% fetal bovine serum (FBS), penicillin, streptomycin, and fungizone.
AsPC-1 and 22Rv-1 cells were maintained in RPMI1640 supplemented with 10%
fetal bovine serum (FBS), penicillin, streptomycin, and fungizone.
HPAF-II cells were maintained in MEM supplemented with 10% fetal bovine
serum (FBS), penicillin, streptomycin, and fungizone. For the MTS assay,
cells were plated in 96-well plates at 2,000 cells per well and incubated
with various doses of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione in combination with gemcitibine, docetaxel or
carboplatin for 72 hr. The layout of dose range of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione, gemcitibine, docetaxel and carboplatin for
drug combination analysis was determined MTS was added to each well and
plates were incubated for 4 hr at 37° C. The absorbance of each
well was measured at 492 nm using a microplate reader. Combination
indexes of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with gemcitibine, docetaxel or carboplatin
among the various cell lines was determined by CalcuSyn® (Biosoft).

[0373]Combination indices of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione with gemcitabine, Docetaxel, and carboplatin in
various cell lines were analyzed.

[0374]As described in Example 85, the combination of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione and gemcitabine shows a range of synergistic
anti-proliferative effects in three of five human pancreatic cancer
lines.

[0375]The combination of
(-)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-
-yl)pyrrolidine-2,5,-dione and Docetaxel demonstrates a range of
synergistic anti-proliferative effects in two of three human prostate
cancer lines, i.e. 22RV1 and DU145 as shown in Table 17.